Dominik Niopek

Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells

The function of many eukaryotic proteins is regulated by highly dynamic changes in their nucleocytoplasmic distribution. The ability to precisely and reversibly control nuclear translocation would, therefore, allow dissecting and engineering cellular networks. Here we develop a genetically encoded, light-inducible nuclear localization signal (LINuS) based on the LOV2 domain of Avena sativa phototropin 1. LINuS is a small, versatile tag, customizable for different proteins and cell types. LINuS-mediated nuclear import is fast and reversible, and can be tuned at different levels, for instance, by introducing mutations that alter AsLOV2 domain photo-caging properties or by selecting nuclear localization signals (NLSs) of various strengths. We demonstrate the utility of LINuS in mammalian cells by controlling gene expression and entry into mitosis with blue light.

Optogenetic control of nuclear protein export

Active nucleocytoplasmic transport is a key mechanism underlying protein regulation in eukaryotes. While nuclear protein import can be controlled in space and time with a portfolio of optogenetic tools, protein export has not been tackled so far. Here we present a light-inducible nuclear export system (LEXY) based on a single, genetically encoded tag, which enables precise spatiotemporal control over the export of tagged proteins. A constitutively nuclear, chromatin-anchored LEXY variant expands the method towards light inhibition of endogenous protein export by sequestering cellular CRM1 receptors. We showcase the utility of LEXY for cell biology applications by regulating a synthetic repressor as well as human p53 transcriptional activity with light. LEXY is a powerful addition to the optogenetic toolbox, allowing various novel applications in synthetic and cell biology.

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.

Creating functional engineered variants of the single-module non-ribosomal peptide synthetase IndC by T domain exchange

Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties(1). These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors(2). A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol(1,2). Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling(3), i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically >80% for most AAV serotypes), or peptide display(4,5), i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Optogenetic Control of Nuclear Protein Import in Living Cells Using Light‐Inducible Nuclear Localization Signals (LINuS)

Many biological processes are regulated by the timely import of specific proteins into the nucleus. The ability to spatiotemporally control the nuclear import of proteins of interest therefore allows study of their role in a given biological process as well as controlling this process in space and time. The light‐inducible nuclear localization signal (LINuS) was developed based on a natural plant photoreceptor that reversibly triggers the import of proteins of interest into the nucleus with blue light. Each LINuS is a small, genetically encoded domain that is fused to the protein of interest at the N or C terminus. These protocols describe how to carry out initial microscopy‐based screening to assess which LINuS variant works best with a protein of interest.

To go, or not to go, that is the question - six personal reflections on how geographic mobility may affect your career and life.

Sooner or later, every aspiring scientist faces the question whether to trade daily routine and familiar environment for a shortor long-term venture into the big unknown in order to continue studying or working abroad. As the final choice will invoke dramatic changes not only for one’s own career and life, but also for family and friends, making the ‘‘right’’ decision is far from easy. Here, two ambitious young Master’s students who are in this situation describe their personal uncertainties and doubts, but also their hopes and dreams concerning their next steps. In response, two PhD students and two junior group leaders summarize their experiences of how geographic mobility has affected – positively or negatively – their own careers and lives, hoping this will not only aid the Master’s students but every reader struggling with the fundamental decision to leave or stay.

Controlling Cells with Light and LOV

Optogenetics is a powerful method for studying dynamic processes in living cells and has advanced cell biology research over the recent past. Key to the successful application of optogenetics is the careful design of the light‐sensing module, typically employing a natural or engineered photoreceptor that links the exogenous light input to the cellular process under investigation. Light–oxygen–voltage (LOV) domains, a highly diverse class of small blue light sensors, have proven to be particularly versatile for engineering optogenetic input modules. These can function via diverse modalities, including inducible allostery, protein recruitment, dimerization, or dissociation. This study reviews recent advances in the development of LOV domain‐based optogenetic tools and their application for studying and controlling selected cellular functions. Focusing on the widely employed LOV2 domain from Avena sativa phototropin‐1, this review highlights the broad spectrum of engineering opportunities that can be explored to achieve customized optogenetic regulation. Finally, major bottlenecks in the development of optogenetic methods are discussed and strategies to overcome these with recent synthetic biology approaches are pointed out.

Engineered anti-CRISPR proteins for optogenetic control of CRISPR–Cas9

Anti-CRISPR proteins are powerful tools for CRISPR–Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR–Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.CASANOVA uses LOV and blue light to regulate CRISPR activity.

AAVvector-mediated in vivo reprogramming into pluripotency

In vivo reprogramming of somatic cells into induced pluripotent stem cells (iPSC) holds vast potential for basic research and regenerative medicine. However, it remains hampered by a need for vectors to express reprogramming factors (Oct-3/4, Klf4, Sox2, c-Myc; OKSM) in selected organs. Here, we report OKSM delivery vectors based on pseudotyped Adeno-associated virus (AAV). Using the AAV-DJ capsid, we could robustly reprogram mouse embryonic fibroblasts with low vector doses. Swapping to AAV8 permitted to efficiently reprogram somatic cells in adult mice by intravenous vector delivery, evidenced by hepatic or extra-hepatic teratomas and iPSC in the blood. Notably, we accomplished full in vivo reprogramming without c-Myc. Most iPSC generated in vitro or in vivo showed transcriptionally silent, intronic or intergenic vector integration, likely reflecting the increased host genome accessibility during reprogramming. Our approach crucially advances in vivo reprogramming technology, and concurrently facilitates investigations into the mechanisms and consequences of AAV persistence.In vivo reprogramming of somatic cells is hampered by the need for vectors to express the OKSM factors in selected organs. Here the authors report new AAV-based vectors capable of in vivo reprogramming at low doses.