Tailoring Cardiac Synthetic Transcriptional Modulation Towards Precision Medicine

Molecular and genetic differences between individual cells within tissues underlie cellular heterogeneities defining organ physiology and function in homeostasis as well as in disease states. Transcriptional control of endogenous gene expression has been intensively studied for decades. Thanks to a fast-developing field of single cell genomics, we are facing an unprecedented leap in information available pertaining organ biology offering a comprehensive overview. The single-cell technologies that arose aided in resolving the precise cellular composition of many organ systems in the past years. Importantly, when applied to diseased tissues, the novel approaches have been immensely improving our understanding of the underlying pathophysiology of common human diseases. With this information, precise prediction of regulatory elements controlling gene expression upon perturbations in a given cell type or a specific context will be realistic. Simultaneously, the technological advances in CRISPR-mediated regulation of gene transcription as well as their application in the context of epigenome modulation, have opened up novel avenues for targeted therapy and personalized medicine. Here, we discuss the fast-paced advancements during the recent years and the applications thereof in the context of cardiac biology and common cardiac disease. The combination of single cell technologies and the deep knowledge of fundamental biology of the diseased heart together with the CRISPR-mediated modulation of gene regulatory networks will be instrumental in tailoring the right strategies for personalized and precision medicine in the near future. In this review, we provide a brief overview of how single cell transcriptomics has advanced our knowledge and paved the way for emerging CRISPR/Cas9-technologies in clinical applications in cardiac biomedicine.

Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

Conditional gene expression is a powerful tool for genetic analysis of biological phenomena. In the widely used “lox-stop-lox” approach, insertion of a stop cassette consisting of a series of stop codons and polyadenylation signals flanked by lox sites into the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been successfully used in many experimental systems, this design has certain limitations. Here, we describe the Floxed exon (Flexon) approach, which uses a stop cassette composed of an artificial exon flanked by artificial introns, designed to cause premature termination of translation and nonsense-mediated decay of the mRNA and allowing for flexible placement into a gene. We demonstrate its efficacy in Caenorhabditis elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression of green fluorescent protein (GFP) is obtained in specific lineages. We also demonstrate its efficacy in an endogenous gene context: we inserted a flexon into the Argonaute gene rde-1 to abrogate RNA interference (RNAi), and restored RNAi tissue specifically by expression of Cre. Finally, we describe several potential additional applications of the Flexon approach, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation, and generation of genetic mosaics. The Flexon approach should be feasible in any system where a site-specific recombination-based method may be applied.

OperonSEQer: A set of machine-learning algorithms with threshold voting for detection of operon pairs using short-read RNA-sequencing data

Operon prediction in prokaryotes is critical not only for understanding the regulation of endogenous gene expression, but also for exogenous targeting of genes using newly developed tools such as CRISPR-based gene modulation. A number of methods have used transcriptomics data to predict operons, based on the premise that contiguous genes in an operon will be expressed at similar levels. While promising results have been observed using these methods, most of them do not address uncertainty caused by technical variability between experiments, which is especially relevant when the amount of data available is small. In addition, many existing methods do not provide the flexibility to determine the stringency with which genes should be evaluated for being in an operon pair. We present OperonSEQer, a set of machine learning algorithms that uses the statistic and p-value from a non-parametric analysis of variance test (Kruskal-Wallis) to determine the likelihood that two adjacent genes are expressed from the same RNA molecule. We implement a voting system to allow users to choose the stringency of operon calls depending on whether your priority is high recall or high specificity. In addition, we provide the code so that users can retrain the algorithm and re-establish hyperparameters based on any data they choose, allowing for this method to be expanded as additional data is generated. We show that our approach detects operon pairs that are missed by current methods by comparing our predictions to publicly available long-read sequencing data. OperonSEQer therefore improves on existing methods in terms of accuracy, flexibility, and adaptability.

N6-Methyladenosine Regulatory Machinery is Involved In Viral Infection of Plum Pox Virus And Potato Virus Y In Nicotiana Benthamiana

Abstract N6-methyladenosine (m6A) is a post-transcriptional modification of biological mRNA and non-coding RNAs, which by regulating the mRNA stability and translation. It has been demonstrated that m6A methylation has a regulatory effect on human RNA virus replication. In this project, Plum pox virus (PPV) and Potato Y virus (PVY) were used to examine the m6A modification in Nicotiana benthamiana during natural infection. The results showed that the global level of m6A in both PVY and PPV infected plants were significantly decreased than non-infected plants. Particularly, the PPV and PVY infection could alter the m6A level of the host endogenous gene. This is suggesting that plant viruses may disrupt the balance of the m6A in plant. Meanwhile, we found that viral genome RNA can be targeted by m6A methylation. Two m6A-enrich regions in PPV genome RNA and four in PVY genome RNA were detected, which are located in the coding region of viruses. Based on the ALKB and METTL sequences in the transcriptome sequencing data of the virus-infected plant, we cloned 2 NbALKB genes and 2 NbMETTL genes in N. benthamiana . According to results of transient expression and VIGS assay, NbALKB gene appears slightly contributing PPV and PVY infection. NbMETTL gene showed certain inhibition effect in PPV infection, but not PVY. Therefore, our data suggested that m6A methylation in plant might be an anti-viral strategy in some plant viruses.

DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

CRISPR technology has made generation of gene knockouts widely achievable in cells. However, once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon), approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene transcription with the ability to reactivate transcription on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, expression kinetics and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in cells.GRAPHICAL ABSTRACTIN BRIEFWe describe development of DExCon, LUXon and DExogron approaches, where a single CRIPR/Cas9-mediated gene editing event can block endogenous gene expression, with the ability to reactivate expression encoded such that even silent genes can be expressed. Expression can be controlled systematically using doxycycline, or spatiotemporally by light, allowing fluorescent tagging of endogenous proteins and quantification of expression kinetics, protein dynamics and stability for highly similar genes such as members of the Rab11 family.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Evaluation of the OsTIR1 and AtAFB2 AID Systems for Genome Architectural Protein Degradation in Mammalian Cells

The auxin-inducible degron (AID) system is a promising tool for dynamic protein degradation. In mammalian cells, this approach has become indispensable to study fundamental molecular functions, such as replication, chromatin dynamics, or transcription, which are otherwise difficult to dissect. We present evaluation of the two prominent AID systems based on OsTIR1 and AtAFB2 auxin receptor F-box proteins (AFBs). We analyzed degradation dynamics of cohesin/condensin complex subunits in mouse embryonic stem cells (Rad21, Smc2, Ncaph, and Ncaph2) and human haploid HAP1 line (RAD21, SMC2). Double antibiotic selection helped achieve high homozygous AID tagging of an endogenous gene for all genes using CRISPR/Cas9. We found that the main challenge for successful protein degradation is obtaining cell clones with high and stable AFB expression levels due to the mosaic expression of AFBs. AFB expression from a transgene tends to decline with passages in the absence of constant antibiotic selection, preventing epigenetic silencing of a transgene, even at the AAVS1 safe-harbor locus. Comparing two AFBs, we found that the OsTIR1 system showed weak dynamics of protein degradation. At the same time, the AtAFB2 approach was very efficient even in random integration of AFB-expressed transgenes. Other factors such as degradation dynamics and low basal depletion were also in favor of the AtAFB2 system.

A functional genetic toolbox for human tissue-derived organoids

Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag' our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly-targeted cells. Our toolbox also incorporates conditional gene knock-down or overexpression using tightly-inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many on-going descriptive studies, such as the Human Cell Atlas Project.