Search

Given the large amount of genome-wide data that have been collected during the last decades, a good understanding of how and why cells change during development, homeostasis, and disease might be expected. Unfortunately, the opposite is true; triggers that cause cellular state changes remain elusive, and the underlying molecular mechanisms are poorly understood. Although genes with the potential to influence cell states are known, the historic dependency on methods that manipulate gene expression outside the endogenous chromatin context has prevented us from understanding how cells organize, interpret, and protect cellular programs. Fortunately, recent methodological innovations are now providing options to answer these outstanding questions, by allowing to target and manipulate individual genomic and epigenomic loci. In particular, three experimental approaches are now feasible due to DNA targeting tools, namely, activation and/or repression of master transcription factors in their endogenous chromatin context; targeting transcription factors to endogenous, alternative, or inaccessible sites; and finally, functional manipulation of the chromatin context. In this article, we discuss the molecular basis of DNA targeting tools and review the potential of these new technologies before we summarize how these have already been used for the manipulation of cellular states and hypothesize about future applications.

Summary Astrocyte-to-neuron conversion is a promising avenue for neuronal replacement therapy. Neurons are particularly dependent on mitochondrial function, but how well mitochondria adapt to the new fate is unknown. Here, we determined the comprehensive mitochondrial proteome of cortical astrocytes and neurons, identifying about 150 significantly enriched mitochondrial proteins for each cell type, including transporters, metabolic enzymes, and cell-type-specific antioxidants. Monitoring their transition during reprogramming revealed late and only partial adaptation to the neuronal identity. Early dCas9-mediated activation of genes encoding mitochondrial proteins significantly improved conversion efficiency, particularly for neuron-enriched but not astrocyte-enriched antioxidant proteins. For example, Sod1 not only improves the survival of the converted neurons but also elicits a faster conversion pace, indicating that mitochondrial proteins act as enablers and drivers in this process. Transcriptional engineering of mitochondrial proteins with other functions improved reprogramming as well, demonstrating a broader role of mitochondrial proteins during fate conversion., Graphical abstract, Highlights • Mitochondrial proteomes of cortical astrocytes and neurons are distinct • Astrocyte-enriched mitochondrial proteins are downregulated late in neuronal conversion • Neuron-enriched mitochondrial proteins are upregulated late in neuronal conversion • Early induction of neuronal mitochondrial proteins improves neuronal reprogramming, Russo et al. identify mitochondrial proteins enriched in neurons or astrocytes. Astrocyte-enriched mitochondrial proteins are often only partially downregulated during astrocyte-to-neuron direct reprogramming. Neuron-enriched ones are upregulated late and mainly in reprogrammed neurons. CRISPRa-mediated early induction of neuron-enriched mitochondrial proteins boosts direct neuronal reprogramming speed and efficiency.

Regulation of adult neural stem cell (NSC) number is critical for lifelong neurogenesis. Here, we identified a post-transcriptional control mechanism, centered around the microRNA 204 (miR-204), to control the maintenance of quiescent (q)NSCs. miR-204 regulates a spectrum of transcripts involved in cell cycle regulation, neuronal migration, and differentiation in qNSCs. Importantly, inhibition of miR-204 function reduced the number of qNSCs in the subependymal zone (SEZ) by inducing pre-mature activation and differentiation of NSCs without changing their neurogenic potential. Strikingly, we identified the choroid plexus of the mouse lateral ventricle as the major source of miR-204 that is released into the cerebrospinal fluid to control number of NSCs within the SEZ. Taken together, our results describe a novel mechanism to maintain adult somatic stem cells by a niche-specific miRNA repressing activation and differentiation of stem cells.

Master transcription factors have the ability to direct and reverse cellular identities, and consequently their genes must be subject to particular transcriptional control. However, it is unclear which molecular processes are responsible for impeding their activation and safeguarding cellular identities. Here we show that the targeting of dCas9-VP64 to the promoter of the master transcription factor Sox1 results in strong transcript and protein up-regulation in neural progenitor cells (NPCs). This gene activation restores lost neuronal differentiation potential, which substantiates the role of Sox1 as a master transcription factor. However, despite efficient transactivator binding, major proportions of progenitor cells are unresponsive to the transactivating stimulus. By combining the transactivation domain with epigenome editing we find that among a series of euchromatic processes, the removal of DNA methylation (by dCas9-Tet1) has the highest potential to increase the proportion of cells activating foreign master transcription factors and thus breaking down cell identity barriers., Master transcription factors dominantly direct cell fate and barriers ensuring their tissue specific silencing are not clearly defined. Here, the authors demonstrate that inefficient targeted transactivation of Sox1 in neural progenitor cells is surmountable through targeted promoter demethylation using dCas9-Tet1.

Novel applications based on the bacterial CRISPR system make genetic, genomic, transcriptional and epigenomic engineering widely accessible for the first time. A significant advantage of CRISPR over previous methods is its tremendous adaptability due to its bipartite nature. Cas9 or its engineered variants define the molecular effect, while short gRNAs determine the targeting sites. A majority of CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into single cells, either as an essential precondition, to increase responsive cell populations or to enhance phenotypic outcomes. Despite these requirements, methods allowing the efficient generation and delivery of multiple gRNA expression units into single cells are still sparse. Here we present STAgR (String assembly gRNA cloning), a single step gRNA multiplexing system, that obtains its advantages by employing the N20 targeting sequences as necessary homologies for Gibson assembly. We show that STAgR allows reliable and cost-effective generation of vectors with high numbers of gRNAs enabling multiplexed CRISPR approaches. Moreover, STAgR is easily customizable, as vector backbones as well as gRNA structures, numbers and promoters can be freely chosen and combined. Finally, we demonstrate STAgR's widespread functionality, its efficiency in multi-targeting approaches, using it for both, genome and transcriptome editing, as well as applying it in vitro and in vivo.

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

Summary: Zebrafish have a high capacity to replace lost neurons after brain injury. New neurons involved in repair are generated by a specific set of glial cells, known as ependymoglial cells. We analyze changes in the transcriptome of ependymoglial cells and their progeny after injury to infer the molecular pathways governing restorative neurogenesis. We identify the aryl hydrocarbon receptor (AhR) as a regulator of ependymoglia differentiation toward post-mitotic neurons. In vivo imaging shows that high AhR signaling promotes the direct conversion of a specific subset of ependymoglia into post-mitotic neurons, while low AhR signaling promotes ependymoglial proliferation. Interestingly, we observe the inactivation of AhR signaling shortly after injury followed by a return to the basal levels 7 days post injury. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Taken together, we identify AhR signaling as a crucial regulator of restorative neurogenesis timing in the zebrafish brain. : Zebrafish have a high capacity to replace lost neurons after brain injury. Di Giaimo et al. identify the aryl hydrocarbon receptor (AhR) as a crucial regulator of restorative neurogenesis timing in the zebrafish brain. Interference with timely AhR regulation after injury leads to aberrant restorative neurogenesis. Keywords: neurogenesis, regeneration, aryl hydrocarbon receptor, direct conversion, zebrafish, live imaging

Background The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. Results Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. Conclusions The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3268-z) contains supplementary material, which is available to authorized users.