Increased expression of vimentin intermediate filaments (VIFs) enhances directed cell migration, but the mechanism behind VIFs’ effect on motility is not understood. VIFs interact with microtubules, ...whose organization contributes to polarity maintenance in migrating cells. Here, we characterize the dynamic coordination of VIF and microtubule networks in wounded monolayers of retinal pigment epithelial cells. By genome editing, we fluorescently labeled endogenous vimentin and α-tubulin, and we developed computational image analysis to delineate architecture and interactions of the two networks. Our results show that VIFs assemble an ultrastructural copy of the previously polarized microtubule network. Because the VIF network is long-lived compared to the microtubule network, VIFs template future microtubule growth along previous microtubule tracks, thus providing a feedback mechanism that maintains cell polarity. VIF knockdown prevents cells from polarizing and migrating properly during wound healing. We suggest that VIFs’ templating function establishes a memory in microtubule organization that enhances persistence in cell polarization in general and migration in particular.
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•Endogenous vimentin and α-tubulin were fluorescently labeled by genome editing•Vimentin-microtubule interactions were analyzed by quantitative live-cell imaging•Vimentin is a template for microtubule organization•Vimentin’s templating function stabilizes cell polarity during directed migration
Quantitative live-cell imaging of genome-edited cells shows that, during directed cell migration, vimentin intermediate filaments assemble in an ultrastructural copy of the polarized microtubule network. Because vimentin turns over slower than microtubules, it stabilizes and templates microtubule organization, thus providing persistence to existing cell polarity.
Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding ...domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC.
Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach ...can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.
Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical ...association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.
Prostate cancer is the second leading cause of male cancer deaths due to disease progression to castration-resistant prostate cancer (CRPC). Androgen receptor (AR) splice variants including AR-V7 ...function as constitutively active transcription factors in CRPC cells, thereby promoting resistance to AR-targeted therapies. To date, there are no AR variant-specific treatments for CRPC. Here we report that the splicing of AR variants AR-V7 as well as AR-V1 and AR-V9 is regulated coordinately by a single polyadenylation signal in AR intron 3. Blocking this signal with morpholino technology or silencing of the polyadenylation factor CPSF1 caused a splice switch that inhibited expression of AR variants and blocked androgen-independent growth of CRPC cells. Our findings support the development of new therapies targeting the polyadenylation signal in AR intron 3 as a strategy to prevent expression of a broad array of AR variants in CRPC.
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Bioluminescence is a powerful biological signal that scientists have repurposed as a reporter for gene expression in plants and animals. However, there are downsides associated with the need to ...provide a substrate to these reporters, including its high cost and non-uniform tissue penetration. In this work we reconstitute a fungal bioluminescence pathway (FBP)
using a composable toolbox of parts. We demonstrate that the FBP can create luminescence across various tissues in a broad range of plants without external substrate addition. We also show how our toolbox can be used to deploy the FBP
to build auto-luminescent reporters for the study of gene-expression and hormone fluxes. A low-cost imaging platform for gene expression profiling is also described. These experiments lay the groundwork for future construction of programmable auto-luminescent plant traits, such as light driven plant-pollinator interactions or light emitting plant-based sensors.
Dear Editor 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 nucleaseencoding DNA into the host genome. Random integration of DNA is a particular concern for trait improvement and gene function studies, because it may lead to unintended gene inactivation or it may alter expression of host genes. Furthermore, for crop varieties created using biotechnology, the presence of foreign DNA is a trigger for regulation by many governmental agencies （Voytas and Gao, 2014）. Therefore, methods to modify plant genomes that do not require DNA delivery would have value in both commercial and academic settings. Here we demonstrate nontransgenic plant genome engineering by introducing sequencespecific nucleases as purified protein. This approach enabled targeted mutagenesis of endogenous sequences within plant cells, while avoiding integration of foreign DNA into the genome.
Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques ...or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.
Genome-editing has revolutionized biology. When coupled with a recently streamlined regulatory process by the U.S. Department of Agriculture and the potential to generate transgene-free varieties, ...genome-editing provides a new avenue for crop improvement. For heterozygous, polyploid and vegetatively propagated crops such as cultivated potato,
Group Tuberosum L., genome-editing presents tremendous opportunities for trait improvement. In potato, traits such as improved resistance to cold-induced sweetening, processing efficiency, herbicide tolerance, modified starch quality and self-incompatibility have been targeted utilizing CRISPR/Cas9 and TALEN reagents in diploid and tetraploid clones. However, limited progress has been made in other such crops including sweetpotato, strawberry, grapes, citrus, banana etc., In this review we summarize the developments in genome-editing platforms, delivery mechanisms applicable to plants and then discuss the recent developments in regulation of genome-edited crops in the United States and The European Union. Next, we provide insight into the challenges of genome-editing in clonally propagated polyploid crops, their current status for trait improvement with future prospects focused on potato, a global food security crop.
The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become ...important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths - from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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