RTA and LANA Competitively Regulate let-7a/RBPJ Signal to Control KSHV Replication

The recombination signal binding protein for immunoglobulin kappa J region (RBPJ) has a dual effect on Kaposi’s sarcoma-associated herpesvirus (KSHV) replication. RBPJ interaction with replication and transcription activator (RTA) is essential for lytic replication, while the interaction with latency-associated nuclear antigen (LANA) facilitates latent infection. Furthermore, our previous study found that LANA decreased RBPJ through upregulating miRNA let-7a. However, it is unclear whether RTA regulates the expression of RBPJ. Here, we show RTA increases RBPJ by decreasing let-7a. During KSHV replication, the RBPJ expression level was positively correlated with the RTA expression level and negatively correlated with the LANA expression level. The let-7a expression level was inverse to RBPJ. Knockdown of RBPJ inhibited the self-activation of RTA promoter and LANA promoter and weakened LANA’s inhibition of RTA promoter. Collectively, these findings indicate that RTA and LANA compete for let-7a/RBPJ signal to control the KSHV replication. Regulating the RBPJ expression level by RTA and LANA plays an important role during KSHV replication.

XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks

Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.

A Targeted In-Fusion Expression System for Recombinant Protein Production in Bombyx mori

The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.

Targeted deletion of liver-expressed Choriogenin L results in the production of soft eggs and infertility in medaka, Oryzias latipes

Egg envelopes (chorions) in medaka, Oryzias latipes, are composed of three major glycoproteins: ZI-1, − 2, and − 3. These gene-encoded chorion glycoproteins are expressed in the liver and/or ovarian oocytes of sexually mature female fish. In medaka, the glycoproteins produced in the female liver are induced by estrogen as Choriogenin (Chg.) H and Chg. H minor (m), which correspond to the zona pellucida (ZP) B (ZPB) protein in mammals, and Chg. L, which corresponds to ZPC in mammals. Chg. H, Chg. Hm, and Chg. L, are then converted to ZI-1, − 2, and − 3, respectively, during oogenesis in medaka ovaries.In the present study, we established a medaka line in which the chg.l gene was inactivated using the transcription activator-like effector nuclease (TALEN) technique. Neither intact chg.l transcripts nor Chg. L proteins were detected in livers of sexually mature female homozygotes for the mutation (homozygous chg.l knockout: chg.l−/−). The chg.l−/− females spawned string-like materials containing “smashed eggs.” Closer examination revealed the oocytes in the ovaries of chg.l−/− females had thin chorions, particularly at the inner layer, despite a normal growth rate. In comparing chorions from normal (chg.l+/+) and chg.l−/− oocytes, the latter exhibited abnormal architecture in the chorion pore canals through which the oocyte microvilli pass. These microvilli mediate the nutritional exchange between the oocyte and surrounding spaces and promote sperm-egg interactions during fertilization. Thus, following in vitro fertilization, no embryos developed in the artificially inseminated oocytes isolated from chg.l−/− ovaries. These results demonstrated that medaka ZI-3 (Chg.L) is the major component of the inner layer of the chorion, as it supports and maintains the oocyte’s structural shape, enabling it to withstand the pressures exerted against the chorion during spawning, and is essential for successful fertilization. Therefore, gene products of oocyte-specific ZP genes that may be expressed in medaka oocytes cannot compensate for the loss Chg. L function to produce offspring for this species.

Improving disease resistance in plants by editing the epigenome

Pathogens rely on expression of host susceptibility (S) genes to promote infection and disease. As DNA methylation is an epigenetic modification that affects gene expression, blocking access to S genes through targeted methylation could increase disease resistance. Xanthomonas axonopodis pv. manihotis, the causal agent of cassava bacterial blight (CBB), uses transcription activator-like20 (TAL20) to induce expression of the S gene MeSWEET10a. We directed methylation to the TAL20 effector binding element within the MeSWEET10a promoter using a synthetic zinc-finger DNA binding domain fused to a component of the RNA-directed DNA methylation pathway. We demonstrate that this methylation prevents TAL20 binding, blocks transcriptional activation of MeSWEET10a in vivo and that these plants display increased resistance to CBB while maintaining normal growth and development. This work establishes epigenome editing as a new strategy for crop improvement.

Tal2c Activates the Expression of OsF3H04g to Promote Infection as a Redundant TALE of Tal2b in Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae delivers transcription activator-like effectors (TALEs) into plant cells to facilitate infection. Following economic principles, the redundant TALEs are rarely identified in Xanthomonas. Previously, we identified the Tal2b, which activates the expression of the rice 2-oxoglutarate-dependent dioxygenase gene OsF3H03g to promote infection in the highly virulent strain of X. oryzae pv. oryzicola HGA4. Here, we reveal that another clustered TALE, Tal2c, also functioned as a virulence factor to target rice OsF3H04g, a homologue of OsF3H03g. Transferring Tal2c into RS105 induced expression of OsF3H04g to coincide with increased susceptibility in rice. Overexpressing OsF3H04g caused higher susceptibility and less salicylic acid (SA) production compared to wild-type plants. Moreover, CRISPR–Cas9 system-mediated editing of the effector-binding element in the promoters of OsF3H03g or OsF3H04g was found to specifically enhance resistance to Tal2b- or Tal2c-transferring strains, but had no effect on resistance to either RS105 or HGA4. Furthermore, transcriptome analysis revealed that several reported SA-related and defense-related genes commonly altered expression in OsF3H04g overexpression line compared with those identified in OsF3H03g overexpression line. Overall, our results reveal a functional redundancy mechanism of pathogenic virulence in Xoc in which tandem Tal2b and Tal2c specifically target homologues of host genes to interfere with rice immunity by reducing SA.

TALEN-Mediated Gene Editing of slc24a5 (Solute Carrier Family 24, Member 5) in Kawakawa, Euthynnus affinis

Transcription activator-like effector (TALE) nucleases (TALENs) mediated gene editing methods are becoming popular and have revealed the staggering complexity of genome control during development. Here, we present a simple and efficient gene knockout using TALENs in kawakawa, Euthynnus affinis, using slc24a5. We examined slc24a5 gene expression and functional differences between two TALENs that hold the TALE scaffolds, +153/+47 and +136/+63 and target slc24a5. Developmental changes in slc24a5 transcripts were seen in early-stage embryos by real-time PCR; slc24a5 expression was first detected 48 h post fertilization (hpf), which increased dramatically at 72 hpf. Four TALENs, 47- and 63-type of two different target loci (A and B), respectively, were constructed using Platinum TALEN and evaluated in vitro by a single-strand annealing (SSA) assay. TALEN activities were further evaluated in vivo by injecting TALEN mRNAs in the two-cell stage of the zygote. Most of the TALEN-induced mutants showed mosaic patterns in the retinal pigment epithelium (RPE) and fewer melanin pigments on the body at 72 hpf and later when compared to the control, implying the gene’s association with melanin pigment formation. A heteroduplex mobility assay (HMA) and the genome sequence further confirmed the TALEN-induced mutations of substitution, insertion, and deletion at an endogenous locus.

Genome editing in cultured fishes

With external fertilization, high fecundity, and established methods for propagation and larval rearing for cultured species, fish provide systems well suited to genome-editing procedures. While early experiments utilized zinc-finger nucleases and transcription activator-like effector nucleases (TALENs), most recent ones have used the CRISPR/Cas9 editor, and achieved rates of targeted genomic insertion well above those of classical transgenic methods, with lower frequencies of off-site integration. Genome-editing experiments with cultured fishes have focused on improving growth rate and disease resistance, achievement of reproductive confinement, and other valued traits. As reviewed here, advances in knowledge of key molecular pathways and, in some cases, favorable alterations of phenotype have been achieved. For example, loss-of-function of myostatin, a negative regulator of muscle growth, led to increased muscle mass, greater weight, and greater fillet yield in genome-edited lines of red sea bream, tiger puffer, and Nile tilapia than in their unedited counterparts. The red sea bream line become the first genome-edited animal to reach commercial production. As for all animals, wide adoption of genome-edited fishes will depend upon addressing issues of regulation, consumer acceptance, and breeding infrastructure.

Epigenetic features improve TALE target prediction

Background The yield of many crop plants can be substantially reduced by plant-pathogenic Xanthomonas bacteria. The infection strategy of many Xanthomonas strains is based on transcription activator-like effectors (TALEs), which are secreted into the host cells and act as transcriptional activators of plant genes that are beneficial for the bacteria.The modular DNA binding domain of TALEs contains tandem repeats, each comprising two hyper-variable amino acids. These repeat-variable diresidues (RVDs) bind to their target box and determine the specificity of a TALE.All available tools for the prediction of TALE targets within the host plant suffer from many false positives. In this paper we propose a strategy to improve prediction accuracy by considering the epigenetic state of the host plant genome in the region of the target box. Results To this end, we extend our previously published tool PrediTALE by considering two epigenetic features: (i) chromatin accessibility of potentially bound regions and (ii) DNA methylation of cytosines within target boxes. Here, we determine the epigenetic features from publicly available DNase-seq, ATAC-seq, and WGBS data in rice.We benchmark the utility of both epigenetic features separately and in combination, deriving ground-truth from RNA-seq data of infections studies in rice. We find an improvement for each individual epigenetic feature, but especially the combination of both.Having established an advantage in TALE target predicting considering epigenetic features, we use these data for promoterome and genome-wide scans by our new tool EpiTALE, leading to several novel putative virulence targets. Conclusions Our results suggest that it would be worthwhile to collect condition-specific chromatin accessibility data and methylation information when studying putative virulence targets of Xanthomonas TALEs.