Rasmus O. Bak

Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34+ hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication

Significance HIV-1 is a lentivirus and replicates through a replication cycle involving several DNA forms including ssDNA. Here we report that synthetic DNA oligos corresponding to DNA forms of the lentivirus replication cycle as well as viral DNA are detected by the immunological DNA sensor IFN-inducible protein 16 (IFI16) and stimulate innate immune responses through a pathway dependent on stimulator of IFN genes (STING). Moreover, we show that replication of HIV-1 is elevated in cells with decreased expression of IFI16 or STING. We suggest IFI16 is a sensor for lentivirus DNA in macrophages stimulating innate immune responses, which contribute to early control of the virus. Replication of lentiviruses generates different DNA forms, including RNA:DNA hybrids, ssDNA, and dsDNA. Nucleic acids stimulate innate immune responses, and pattern recognition receptors detecting dsDNA have been identified. However, sensors for ssDNA have not been reported, and the ability of RNA:DNA hybrids to stimulate innate immune responses is controversial. Using ssDNAs derived from HIV-1 proviral DNA, we report that this DNA form potently induces the expression of IFNs in primary human macrophages. This response was stimulated by stem regions in the DNA structure and was dependent on IFN-inducible protein 16 (IFI16), which bound immunostimulatory DNA directly and activated the stimulator of IFN genes –TANK-binding kinase 1 - IFN regulatory factors 3/7 (STING–TBK1–IRF3/7) pathway. Importantly, IFI16 colocalized and associated with lentiviral DNA in the cytoplasm in macrophages, and IFI16 knockdown in this cell type augmented lentiviral transduction and also HIV-1 replication. Thus, IFI16 is a sensor for DNA forms produced during the lentiviral replication cycle and regulates HIV-1 replication in macrophages.

CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells

The β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia, are caused by mutations in the β-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure β-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult β-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for β-haemoglobinopathies.

miRNA sponges: soaking up miRNAs for regulation of gene expression

MicroRNAs (miRNAs) are small regulatory RNAs that act in an entangled web of interactions with target mRNAs to shape the cellular protein landscape by post‐transcriptional control of mRNA decay and translation. miRNAs are themselves subject to numerous regulatory mechanisms that adjust their prevalence and activity. Emerging evidence suggests that miRNAs are themselves targeted by regulatory RNA species, and the identification of several classes of noncoding RNA molecules carrying miRNA binding sites has added a new intricate dimension to miRNA regulation. Such miRNA ‘sponges’ bind miRNAs and competitively sequester them from their natural targets. Endogenous miRNA sponges, also termed competing endogenous RNAs (ceRNAs), act to buffer the activity of miRNAs on physiologically relevant targets. This class of sponges includes endogenously transcribed pseudogenes, long noncoding RNAs, and recently discovered circular RNAs and may act in large complex networks in conjunction with miRNAs to regulate the output of protein. With the growing demand of regulating miRNA activity for experimental purposes and potential future clinical use, naturally occurring miRNA sponges are providing inspiration for engineering of gene vector‐encoded sponges as potent inhibitors of miRNA activity. Combined with potent and versatile vector technologies, expression of custom‐designed sponges provides new means of managing miRNAs and soaking up miRNAs for therapeutic regulation of gene expression.

Regulation of pro-inflammatory cytokines TNFα and IL24 by microRNA-203 in primary keratinocytes

Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFα and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFα and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key pro-inflammatory cytokines.

Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses

Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.

Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases

Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in ‘all-in-one’ lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases. DOI: http://dx.doi.org/10.7554/eLife.01911.001

Regulation of cytokines by small RNAs during skin inflammation

Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described.

Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors

MicroRNAs (miRNAs) are ubiquitous regulators of gene expression that contribute to almost any cellular process. Methods for managing of miRNA activity are attracting increasing attention in relation to diverse experimental and therapeutic applications. DNA-encoded miRNA inhibitors expressed from plasmid or virus-based vectors provide persistent miRNA suppression and options of tissue-directed micromanaging. In this report, we explore the potential of exploiting short, hairpin-shaped RNAs for simultaneous suppression of two or more miRNAs. Based on the “Tough Decoy” (TuD) design, we create dual-targeting hairpins carrying two miRNA recognition sites and demonstrate potent co-suppression of different pairs of unrelated miRNAs by a single DNA-encoded inhibitor RNA. In addition, enhanced miRNA suppression is achieved by expression of RNA polymerase II-transcribed inhibitors carrying clustered TuD hairpins with up to a total of eight miRNA recognition sites. Notably, by expressing clustered TuD inhibitors harboring a single recognition site for each of a total of six miRNAs, we document robust parallel suppression of multiple miRNAs by inhibitor RNA molecules encoded by a single expression cassette. These findings unveil a new potential of TuD-based miRNA inhibitors and pave the way for standardizing synchronized suppression of families or clusters of miRNAs.

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.