Perspectives on the Application of Next-generation Sequencing to the Improvement of Africa’s Staple Food Crops

The persistent challenge of insufficient food, unbalanced nutrition, and deteriorating natural resources in the most vulnerable nations, characterized by fast population growth, calls for utilization of innovative technologies to curb constraints of crop production. Enhancing genetic gain by using a multipronged approach that combines conventional and genomic technologies for the development of stress-tolerant varieties with high yield and nutritional quality is necessary. The advent of next-generation sequencing (NGS) technologies holds the potential to dramatically impact the crop improvement process. NGS enables whole-genome sequencing (WGS) and re-sequencing, transcriptome sequencing, metagenomics, as well as high-throughput genotyping, which can be applied for genome selection (GS). It can also be applied to diversity analysis, genetic and epigenetic characterization of germplasm and pathogen detection, identification, and elimination. High-throughput phenotyping, integrated data management, and decision support tools form the necessary supporting environment for effective utilization of genome sequence information. It is important that these opportunities for mainstreaming innovative breeding strategies, enabled by cutting-edge “Omics” technologies, are seized in Africa; however, several constraints must be addressed before the benefit of NGS can be fully realized. African breeding programs must have access to high-throughput genotyping facilities, capacity in the application of genome selection and marker-assisted breeding must be built and supported by capacity in genomic analysis and bioinformatics. This chapter demonstrates how interventions with NGS-enabled innovative strategies can be applied to increase genetic gain with insights from the Consortium of International Agricultural Research (CGIAR) in general and the International Institute of Tropical Agriculture (IITA) in particular

Bioinformatics design of CRISPR guide RNA for genomic knockout of ABCB1 gene

Abstract Background: Over-expression of P-Glycoprotein (Pgp) induces acquired drug resistance. Therefore, targeting Pgp as a dominant efflux transporter involved in emergence of multidrug resistance (MDR) has become a major strategy for reversibility of sensitivity to chemotherapy. Objectives: The aim of this study was to design sgRNAs targeting ABCB1 in order to knockout and inhibit the expression of Pgp in Adriamycin resistant (A2780/ADR) ovarian cancer cell line. Methods: This study was performed as a bioinformatics and computational research in Qazvin University of Medical Sciences in collaboration with the Isfahan University of Medical Sciences during 2015-2016. All the 28 exons of the ABCB1 gene were separately investigated in terms of single guide RNA (sgRNA) target sites with regards to the highest on-target and lowest off-target activities, using www.deskgen.com website. Three sgRNA sequences were chosen and synthesized by the GeneCopoeia company. All the plasmids were validated after extraction using BamH1 and EcoR1 restriction enzymes. Results: Sequences of the three sgRNAs were selected close to the start codon (ATG) in order to maximize the possibility of exons 4 and 5 knockout. Digested pCRISPR-CG01, using BamH1 and EcoR1, was electrophorized on 1.5% agarose gel. Detection of the two 330bp and 10100bp fragments on the gel confirmed the integrity of the plasmid and success of the restriction enzyme digestion. Conclusions: The vectors containing the designed sgRNA sequences and CRISPR associated protein (Cas9) can inhibit Pgp gene expression in cell lines over-expressing this gene, including A2780/ADR. Keywords: Drug Resistance, P-Glycoprotein, Ovarian Cancer, CRISP

Expanding the CRISPR Toolbox in Zebrafish for Studying Development and Disease

The study of model organisms has revolutionized our understanding of the mechanisms underlying normal development, adult homeostasis, and human disease. Much of what we know about gene function in model organisms (and its application to humans) has come from gene knockouts: the ability to show analogous phenotypes upon gene inactivation in animal models. The zebrafish (Danio rerio) has become a popular model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. The RNA-guided CRISPR/Cas9-mediated targeted mutagenesis approaches have provided powerful tools to manipulate the genome toward developing new disease models and understanding the pathophysiology of human diseases. CRISPR-based approaches are being used for the generation of both knockout and knock-in alleles, and also for applications including transcriptional modulation, epigenome editing, live imaging of the genome, and lineage tracing. Currently, substantial effort is being made to improve the specificity of Cas9, and to expand the target coverage of the Cas9 enzymes. Novel types of naturally occurring CRISPR systems [Cas12a (Cpf1); engineered variants of Cas9, such as xCas9 and SpCas9-NG], are being studied and applied to genome editing. Since the majority of pathogenic mutations are single point mutations, development of base editors to convert C:G to T:A or A:T to G:C has further strengthened the CRISPR toolbox. In this review, we provide an overview of the increasing number of novel CRISPR-based tools and approaches, including lineage tracing and base editing

Doing synthetic biology with photosynthetic microorganisms

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.Peer reviewe

Synthetic Genomics

The current advances in sequencing, data mining, DNA synthesis, cloning, in silico modeling, and genome editing have opened a new field of research known as Synthetic Genomics. The main goal of this emerging area is to engineer entire synthetic genomes from scratch using pre-designed building blocks obtained by chemical synthesis and rational design. This has opened the possibility to further improve our understanding of genome fundamentals by considering the effect of the whole biological system on biological function. Moreover, the construction of non-natural biological systems has allowed us to explore novel biological functions so far not discovered in nature. This book summarizes the current state of Synthetic Genomics, providing relevant examples in this emerging field

Dual-active genome-editing reagents

Manipulation of complex genomes has many beneficial downstream applications in agriculture and human gene therapy. Precise genome-editing requires the introduction of a specific DNA double-stand break at a locus of interest, in turn inducing host DNA repair pathways to cause gene knockout through non-homologous end-joining or gene repair using homologous recombination and donor template. No matter the application, the field has depended on a few reagents to introduce precise double-strand breaks in host genomes. LAGLIDADG homing endonucleases or meganucleases harness the natural properties of these rare-cutting enzymes to target precise sequences in a complex genome. Other successful reagents are derived from a type IIS restriction endonuclease, FokI, fused to various DNA-binding architectures such as zinc finger domains and transcription activator-like effector domains. However, the discovery of clustered regularly interspaced short palindromic repeat-associated protein, CRISPR-Cas9, has dominated the field with its ease of design requiring a single RNA molecule to target the sequence of interest. Even with a handful of reagents to choose from, no one reagent is suitable for every application as every reagent has its own set of limitations and advantages. Here we present another potential genome-editing reagent derived from a GIY-YIG homing endonuclease, I-TevI, fused to all four DNA-targeting proteins described above. First, we demonstrate that I-TevI is a portable nuclease domain that can be targeted using Zinc-Fingers and LAGLIDADG proteins. Using these new reagents, we were able to further characterize I-TevI specificity using high throughput in vitro and in vivo screens to highlight important sequence requirements for targeting. Using this knowledge, we systematically engineered new I-TevI variants with altered specificity to broaden the number of targets available for I-TevI-derived reagents. We incorporated these new I-TevI variants into a more versatile dual-active nuclease, TevCas9, capable of introducing two double-strand breaks at a single target site. This dual cleavage event is capable of excising a short DNA fragment from the target site and is unique to I-TevI derived fusions. We envisioned that the monomeric, sequence-specific I-TevI catalytic domain would improve current tools by providing additional specificity and the ability to introduce dual-cleavage event would present unique applications for genome engineering

General Aspects and Applications of Plant Genome Editing: Advancements in Recombinant Protein Production and Viral Vaccine Efficiency through CRISPR/Cas9-Edited N. benthamiana

The implementation of plant-based antigen manufacturing methodologies has accelerated vaccine research by providing a cost-effective, scalable, and safe alternative to traditional protein production systems. This master thesis provides an updated review of recent advances in plant-based protein production platforms, with an emphasis on recombinant proteins generated from CRISPR/Cas9 genome edited Nicotiana benthamiana plants. The first part of this review contains a brief utilization of the CRISPR technology in plant genome engineering, focusing on its advancements and challenges. The thesis includes an overview of various CRISPR applications in plant biology, a detailed assessment of recent advancements in transformation techniques, and an exploration of the challenges associated with sequencing in the context of genome editing. The second part focuses on the use of Nicotiana benthamiana as a model plant to produce recombinant proteins. This review examines the increases in immunogenicity achieved by these unique production platforms through a review of the most recent research. The review also covers the benefits of plant-based protein production platforms, such as cheaper production costs and faster response to emerging risks. The core findings show that plant-based antigen manufacturing methodologies can have an impact on existing protein production systems, potentially leading to the creation of improved vaccines with increased efficacy, safety, and accessibility. The review seeks to offer insights into the current state of research and prospects for using this plant for recombinant protein production, as well as discuss its wider impacts, implications, and future directions for vaccine development

Identification and inactivation of cancer driver mutations using the CRISPR-Cas9 system

Somatische Mutationen sind eine Hauptursache für die Entstehung von Krebs. Allerdings tragen nicht alle Mutationen gleichermaßen zur Tumorentstehung bei. Ein wichtiges Ziel der personalisierten Medizin ist es daher, die für das Wachstum und Überleben des Tumors wesentlichen (sogenannte „Treiber“-Mutationen) von den zahlreichen biologisch neutralen Mutationen (sogenannte „Passagier“-Mutationen) zu unterscheiden. In der vorliegenden Studie etablierte ich einen CRISPR-basierten, genetischen Screen mit dessen Hilfe die funktionelle Rolle von Mutationen bei Krebs untersucht werden kann. Ich konnte nachweißen, dass diese mutationsselektive Strategie geeignet ist, um neue Krebstreibermutationen in der Kolorektalkarzinom- Zelllinie RKO zu identifizieren. Dazu verwendete ich 100 unterschiedliche sgRNAs, welche jeweils eine Krebsmutationssequenz spezifisch schneiden während die Wildtyp-Sequenz nicht verändert wird. Als Kontrolle nutzte ich die Kolorektalkarzinom- Zelllinie HCT116, welche die Zielmutationen nicht trägt. Interessanterweise ergab die Datenanalyse, dass zwei sgRNAs, welche die gleiche Mutation (UTP14A: S99del) schneiden, besonders rasch und ausschließlich in RKO-Zellen verloren gingen. Im Einklang mit den Screening-Ergebnissen führte die individuelle Infektion der Zellen mit diesen sgRNAs zu einem selektiven Verlust in RKO-, nicht aber HCT-Zellen, wodurch UTP14A: S99del als mutmaßliche Treiber-Mutation in RKO-Zellen identifiziert werden konnte. Die weitere Validierung und Charakterisierung dieser mutmaßlichen Treiber-Mutation wird diskutiert. Insgesamt zeigt dieser Ansatz, dass ein solches CRISPR-basiertes System ein leistungsfähiges Werkzeug auch für umfangreichere Untersuchungen von Krebsmutationen darstellt. Parallel dazu setzte ich die CRISPR-Cas-Technologie ein, um bekannte und bisher nicht therapierbare Treiber-Mutationen, wie z.B. innerhalb der Ras-Onkogen-Familie, zu untersuchen. Bemerkenswert ist in diesem Zusammenhang, dass jeder dritte Krebspatient ein durch Mutationen aktiviertes KRAS exprimiert, welches damit das am häufigsten mutierte Onkogen in menschlichen Tumorzellen ist. Im Gegensatz zu anderen Molekülen des MAPK-Signalweges konnte KRAS bisher nicht mittels kleiner, inhibitorischer Moleküle inaktiviert werden. Unter diesen Voraussetzungen birgt ein genomischer, CRISPR-basierter Ansatz das Potenzial, eine dringend benötigte therapeutische Alternative zur KRAS-Inaktivierung zu liefern. Ich entwarf daher drei mutationsselektive sgRNAs abzielend auf die häufigsten KRAS-Mutationen. Obwohl diese Strategie geeignet war, um KRAS-mutierte Tumorzellen in 3 unterschiedlichen Krebszelllinien effizient und spezifisch zu entfernen, führte die langfristige Cas9-Expression zur Bildung von onkogenen, resistenten Klonen. Dieses Phänomen wird durch DNA-Doppelstrangbrüche und die nachfolgend einsetzende, endogene DNA-Reparaturmaschinerie begünstigt. Ich konnte zeigen, dass der Adenin-Basen-Editor im Gegensatz dazu nicht nur in der Lage ist, die KRAS-Mutation ohne Doppelstrangbruch zu inaktivieren, sondern diese auch zur Wildtyp-Sequenz reparieren kann. Mit Hilfe dieses Ansatzes erreichte ich insbesondere bei Vorliegen der G12D-Mutation, einen fast vollständigen Abbau der KRAS-korrigierten Zellen. Die Validierung in patienten-abgeleiteten KRAS-G12D-Organoiden bestätigte die effiziente Korrektur sowie die daraus resultierende erhöhte Sensitivität, wenn auch in einem geringeren Maße als in Zelllinien. Somit konnte in dieser Studie erstmals gezeigt werden, dass Basen-Editierung sowohl in Zelllinien als auch in Organoiden, welche aus Tumorzellen der Patienten stammen, erfolgreich eingesetzt werden kann. Darüber hinaus ist dieses System gut verträglich und induziert weder in Zelllinien noch in Organoiden bei Vorliegen des KRAS Wildtyps unerwünschte Nebeneffekte (sogenannte „Off-target-Effekte“). Langfristig kann die Anwendung von CRISPR-basierten- und Basen-Editierungs-technologien zum Ausschalten von KRAS-Mutationen nicht nur zu einem besseren Verständnis der RAS-Biologie führen, sondern zusammen mit neuen Verabreichungsformen und Technologien die Grundlage für eine dringend benötigte KRAS-Therapie bilden