Transcriptomics-inferred dynamics of SARS-CoV-2 interactions with host epithelial cells.

Virus-host interactions can reveal potentially effective and selective therapeutic targets for treating infection. Here, we performed an integrated analysis of the dynamics of virus replication and the host cell transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using human Caco-2 colon cancer cells as a model. Time-resolved RNA sequencing revealed that, upon infection, cells immediately transcriptionally activated genes associated with inflammatory pathways that mediate the antiviral response, which was followed by an increase in the expression of genes involved in ribosome and mitochondria function, thus suggesting rapid alterations in protein production and cellular energy supply. At later stages, between 24 and 48 hours after infection, the expression of genes involved in metabolic processes-in particular, those related to xenobiotic metabolism-was decreased. Mathematical modeling incorporating SARS-CoV-2 replication suggested that SARS-CoV-2 proteins inhibited the host antiviral response and that virus transcripts exceeded the translation capacity of the host cells. Targeting kinase-dependent pathways that exhibited increases in transcription in host cells was as effective as a virus-targeted inhibitor at repressing viral replication. Our findings in this model system delineate a sequence of SARS-CoV-2 virus-host interactions that may facilitate the identification of druggable host pathways to suppress infection.

Computational design of anti-CRISPR proteins with improved inhibition potency.

Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.

Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.

Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.

The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

A Robust and All-Inclusive Pipeline for Shuffling of Adeno-Associated Viruses.

Adeno-associated viruses (AAV) are attractive templates for engineering of synthetic gene delivery vectors. A particularly powerful technology for breeding of novel vectors with improved properties is DNA family shuffling, i.e., generation of chimeric capsids by homology-driven DNA recombination. Here, to make AAV DNA shuffling available to a wider community, we present a robust experimental and bioinformatical pipeline comprising: (i) standardized and partially codon-optimized plasmids carrying 12 different AAV capsid genes; (ii) a scalable protocol including troubleshooting guide for viral library production; and (iii) the freely available software SALANTO for comprehensive analysis of chimeric AAV DNA and protein sequences. Moreover, we describe a set of 12 premade and ready-to-use AAV libraries. Finally, we demonstrate the usefulness of DNA barcoding technology to trace AAV capsid libraries within a complex mixture. Our protocols and resources facilitate the implementation and tailoring of AAV evolution technology in any laboratory interested in customized viral gene transfer.

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.

A Hepatic GAbp-AMPK Axis Links Inflammatory Signaling to Systemic Vascular Damage.

Increased pro-inflammatory signaling is a hallmark of metabolic dysfunction in obesity and diabetes. Although both inflammatory and energy substrate handling processes represent critical layers of metabolic control, their molecular integration sites remain largely unknown. Here, we identify the heterodimerization interface between the α and ß subunits of transcription factor GA-binding protein (GAbp) as a negative target of tumor necrosis factor alpha (TNF-α) signaling. TNF-α prevented GAbpα and ß complex formation via reactive oxygen species (ROS), leading to the non-energy-dependent transcriptional inactivation of AMP-activated kinase (AMPK) ß1, which was identified as a direct hepatic GAbp target. Impairment of AMPKß1, in turn, elevated downstream cellular cholesterol biosynthesis, and hepatocyte-specific ablation of GAbpα induced systemic hypercholesterolemia and early macro-vascular lesion formation in mice. As GAbpα and AMPKß1 levels were also found to correlate in obese human patients, the ROS-GAbp-AMPK pathway may represent a key component of a hepato-vascular axis in diabetic long-term complications.

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.

CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans.

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.

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.

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.