Crosslinking degree variations enable programming and controlling soft fracture via sideways cracking.

Large deformations of soft materials are customarily associated with strong constitutive and geometrical nonlinearities that originate new modes of fracture. Some isotropic materials can develop strong fracture anisotropy, which manifests as modifications of the crack path. Sideways cracking occurs when the crack deviates to propagate in the loading direction, rather than perpendicular to it.

CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of the maternal-to-zygotic transition in teleosts.

The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT.

In vivo base editing of a pathogenic Eif2b5 variant improves vanishing white matter phenotypes in mice.

Vanishing white matter (VWM) is a fatal leukodystrophy caused by recessive mutations in subunits of the eukaryotic translation initiation factor 2B. Currently, there are no effective therapies for VWM. Here, we assessed the potential of adenine base editing to correct human pathogenic VWM variants in mouse models.

Comparing robotic and manual injection methods in zebrafish embryos for high-throughput RNA silencing using CRISPR-RfxCas13d.

In this study, the authors compared the efficiency of automated robotic and manual injection methods for the CRISPR-RfxCas13d (CasRx) system for mRNA knockdown and Cas9-mediated DNA targeting in zebrafish embryos. They targeted the no tail () gene as a proof-of-principle, evaluating the induced embryonic phenotypes. Both Cas9 and CasRx systems caused loss of function phenotypes for .

Ribonucleotide reductase inhibition improves the symptoms of a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease.

Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes.

The requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAMs, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals.

Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos.

CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our optimized CRISPR-RfxCas13d (CasRx) system.

CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos.

Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines.

Brd4 and P300 Confer Transcriptional Competency during Zygotic Genome Activation.

The awakening of the genome after fertilization is a cornerstone of animal development. However, the mechanisms that activate the silent genome after fertilization are poorly understood. Here, we show that transcriptional competency is regulated by Brd4- and P300-dependent histone acetylation in zebrafish.

RES complex is associated with intron definition and required for zebrafish early embryogenesis.

Pre-mRNA splicing is a critical step of gene expression in eukaryotes. Transcriptome-wide splicing patterns are complex and primarily regulated by a diverse set of recognition elements and associated RNA-binding proteins. The retention and splicing (RES) complex is formed by three different proteins (Bud13p, Pml1p and Snu17p) and is involved in splicing in yeast.

Optimized CRISPR-Cpf1 system for genome editing in zebrafish.

The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37 °C.

CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing.

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus.

MicroRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos.

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families.

Optimization Strategies for the CRISPR-Cas9 Genome-Editing System.

The CRISPR-Cas9 system uncovered in bacteria has emerged as a powerful genome-editing technology in eukaryotic cells. It consists of two components-a single guide RNA (sgRNA) that directs the Cas9 endonuclease to a complementary DNA target site. Efficient targeting of individual genes requires highly active sgRNAs.

Optimized CRISPR-Cas9 System for Genome Editing in Zebrafish.

This protocol describes how to generate and genotype mutants using an optimized CRISPR-Cas9 genome-editing system in zebrafish (CRISPRscan). Because single guide RNAs (sgRNAs) have variable efficiency when targeting specific loci, our protocol starts by explaining how to use the web tool CRISPRscan to design highly efficient sgRNAs. The CRISPRscan algorithm is based on the results of an integrated analysis of more than 1000 sgRNAs in zebrafish, which uncovered highly predictive factors that influence Cas9 activity.

Codon identity regulates mRNA stability and translation efficiency during the maternal-to-zygotic transition.

Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance.

CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.

CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo.

Novel small RNA expression libraries uncover hsa-miR-30b and hsa-miR-30c as important factors in anoikis resistance.

MicroRNAs (miRNAs) have been widely studied in order to elucidate their biological functions. MicroRNA microarrays or miRNA overexpression libraries generated by synthesis and cloning of individual miRNAs have been used to study their different roles. In this work, we have developed a novel methodology to express mature miRNAs and other small RNAs from a double convergent RNA polymerase III promoter.

PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis.

Human securin, also known as human pituitary tumor-transforming gene 1 (pttg1), plays a key role in cell-cycle regulation. Two homologous genes, pttg2 and pttg3, have been identified although very little is known about their physiological function. In this study, we aimed at the characterization of these two pttg1 homologs.

PTTG1/securin modulates microtubule nucleation and cell migration.

Pituitary tumor transforming gene 1 (PTTG1), also known as securin, has been implicated in many biological functions, including inhibition of sister chromatid separation, DNA repair, organ development, and regulation of the expression and secretion of angiogenic and metastatic factors. Although most of these functions of securin seem to depend on the localization of PTTG1 in the nucleus of the cell, a fraction of the protein has been also detected in the cytoplasm. Here we demonstrate that, in different cell types, a portion of cytoplasmic PTTG1 is associated with the cis face of the Golgi apparatus and that this localization depends on PTTG1 phosphorylation status.

Induction of Dlk1 by PTTG1 inhibits adipocyte differentiation and correlates with malignant transformation.

Pituitary tumor-transforming gene-1 (PTTG1) is an oncogene highly expressed in a variety of endocrine, as well as nonendocrine-related cancers. Several tumorigenic mechanisms for PTTG1 have been proposed, one of the best characterized being its capacity to act as a transcriptional activator. To identify novel downstream target genes, we have established cell lines with inducible expression of PTTG1 and a differential display approach to analyze gene expression changes after PTTG1 induction.

pH and Pac1 control development and antifungal activity in Trichoderma harzianum.

In Trichoderma harzianum CECT 2413 external pH regulates essential functions such as growth rate, sporulation, cell and colony morphology, pattern of secreted proteins, gene expression or mycoparasitism-related enzymatic activities. pH regulation is mediated by the transcriptional factor Pac1 (homologous to PacC regulator in other fungi), encoded by pac1 whose expression increases with pH. Two pac1 mutants have been obtained from CECT 2413: P2.

QID74 Cell wall protein of Trichoderma harzianum is involved in cell protection and adherence to hydrophobic surfaces.

Trichoderma is widely used as biocontrol agent against phytopathogenic fungi, and as biofertilizer because of its ability to establish mycorriza-like association with plants. The key factor to the ecological success of this genus is the combination of very active mycoparasitic mechanisms plus effective defense strategies induced in plants. This work, different from most of the studies carried out that address the attacking mechanisms, focuses on elucidating how Trichoderma is able to tolerate hostile conditions.

Glucose uptake in Trichoderma harzianum: role of gtt1.

Using a differential display technique, the gene gtt1, which codes for a high-affinity glucose transporter, has been cloned from the mycoparasite fungus Trichoderma harzianum CECT 2413. The deduced protein sequence of the gtt1 gene shows the 12 transmembrane domains typical of sugar transporters, together with certain residues involved in glucose uptake, such as a conserved arginine between domains IV and V and an aromatic residue (Phe) in the sequence of domain X. The gtt1 gene is transcriptionally regulated, being repressed at high levels of glucose.