Activation of recombinases at specific DNA loci by zinc-finger domain insertions.

Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site.

Measuring university students' ability to recognize argument structures and fallacies.

Theory: Argumentation is crucial for all academic disciplines. Nevertheless, a lack of argumentation skills among students is evident. Two core aspects of argumentation are the recognition of argument structures (e.

Quantification of evolved DNA-editing enzymes at scale with DEQSeq.

We introduce DEQSeq, a nanopore sequencing approach that rationalizes the selection of favorable genome editing enzymes from directed molecular evolution experiments. With the ability to capture full-length sequences, editing efficiencies, and specificities from thousands of evolved enzymes simultaneously, DEQSeq streamlines the process of identifying the most valuable variants for further study and application. We apply DEQSeq to evolved libraries of Cas12f-ABEs and designer-recombinases, identifying variants with improved properties for future applications.

Preschool teachers' pedagogical content knowledge predicts willingness to scaffold early science learning.

Background: The importance of diagnostic and scaffolding activities for early science learning has been shown consistently. However, preschool teachers scarcely engage in them. We developed an instrument to assess preschool teachers' willingness to engage in diagnostic and scaffolding activities in science learning situations and examined its relation with teachers' knowledge, beliefs and practice.

Discovery and characterization of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering.

Tyrosine-type site-specific recombinases (Y-SSRs) are versatile tools for genome engineering due to their ability to mediate excision, integration, inversion and exchange of genomic DNA with single nucleotide precision. The ever-increasing need for sophisticated genome engineering is driving efforts to identify novel SSR systems with intrinsic properties more suitable for particular applications. In this work, we develop a systematic computational workflow for annotation of putative Y-SSR systems and apply this pipeline to identify and characterize eight new naturally occurring Cre-type SSR systems.

Prediction of designer-recombinases for DNA editing with generative deep learning.

Site-specific tyrosine-type recombinases are effective tools for genome engineering, with the first engineered variants having demonstrated therapeutic potential. So far, adaptation to new DNA target site selectivity of designer-recombinases has been achieved mostly through iterative cycles of directed molecular evolution. While effective, directed molecular evolution methods are laborious and time consuming.

Circulating tumour cells in patients with lung cancer universally indicate poor prognosis.

Background: In lung cancer, the relevance of various circulating tumour cell (CTC) subgroups in different lung cancer subtypes is unclear. We performed a comprehensive meta-analysis to assess the prognostic value of CTCs in the different histological types of lung cancer, with particular respect to CTC subtypes, cut-offs and time points of CTC enumeration.

Methods: We searched MEDLINE, Web of Science and Embase alongside relevant studies evaluating the prognostic value of CTCs in lung cancer patients.

The first-in-class ERK inhibitor ulixertinib shows promising activity in mitogen-activated protein kinase (MAPK)-driven pediatric low-grade glioma models.

Background: Pediatric low-grade gliomas (pLGG) are the most common pediatric central nervous system tumors, with driving alterations typically occurring in the MAPK pathway. The ERK1/2 inhibitor ulixertinib (BVD-523) has shown promising responses in adult patients with mitogen-activated protein kinase (MAPK)-driven solid tumors.

Methods: We investigated the antitumoral activity of ulixertinib monotherapy as well as in combination with MEK inhibitors (MEKi), BH3-mimetics, or chemotherapy in pLGG.

Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing.

Unlabelled: KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS.

Rgs16 promotes antitumor CD8 T cell exhaustion.

T cells become functionally exhausted in tumors, limiting T cell-based immunotherapies. Although several transcription factors regulating the exhausted T (T) cell differentiation are known, comparatively little is known about the regulators of T cell survival. Here, we reported that the regulator of G protein signaling 16 (Rgs-16) suppressed T cell survival in tumors.

DNA methylation-independent long-term epigenetic silencing with dCRISPR/Cas9 fusion proteins.

The programmable CRISPR/Cas9 DNA nuclease is a versatile genome editing tool, but it requires the host cell DNA repair machinery to alter genomic sequences. This fact leads to unpredictable changes of the genome at the cut sites. Genome editing tools that can alter the genome without causing DNA double-strand breaks are therefore in high demand.

Functional Therapeutic Target Validation Using Pediatric Zebrafish Xenograft Models.

The survival rate among children with relapsed tumors remains poor, due to tumor heterogeneity, lack of directly actionable tumor drivers and multidrug resistance. Novel personalized medicine approaches tailored to each tumor are urgently needed to improve cancer treatment. Current pediatric precision oncology platforms, such as the INFORM (INdividualized Therapy FOr Relapsed Malignancies in Childhood) study, reveal that molecular profiling of tumor tissue identifies targets associated with clinical benefit in a subgroup of patients only and should be complemented with functional drug testing.

Correction of a Factor VIII genomic inversion with designer-recombinases.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells.

Pairing of single mutations yields obligate Cre-type site-specific recombinases.

Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential.

A large-scale resource for tissue-specific CRISPR mutagenesis in .

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner.

A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus.

Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.

CRISPR/Cas9 as a tool to dissect cancer mutations.

The CRISPR/Cas9 system is transforming many biomedical disciplines, including cancer research. Through its flexible programmability and efficiency to induce DNA double strand breaks it has become straightforward to introduce cancer mutations into cells in vitro and/or in vivo. However, not all mutations contribute equally to tumorigenesis and distinguishing essential mutations for tumor growth and survival from biologically inert mutations is cumbersome.

Scalable, Lightweight, Integrated and Quick-to-Assemble (SLIQ) Hyperdrives for Functional Circuit Dissection.

Independently adjustable multielectrode arrays are routinely used to interrogate neuronal circuit function, enabling chronic monitoring of neuronal ensembles in freely behaving animals at a single-cell, single spike resolution. Despite the importance of this approach, its widespread use is limited by highly specialized design and fabrication methods. To address this, we have developed a Scalable, Lightweight, Integrated and Quick-to-assemble multielectrode array platform.

Prevention of episodic migraines with topiramate: results from a non-interventional study in a general practice setting.

The majority of patients with migraine headaches are treated in non-specialized institutions though data on treatment outcomes are largely derived from tertiary care centers. The current non-interventional study explores efficacy and tolerability outcomes of patients with episodic migraines receiving topiramate as preventive agent in a general practice setting. A total of 366 patients (87% female, mean age 41.