Modern Tools for Genetic Engineering

Site-specific endonucleases create double-strand breaks within the genome and can be targeted to literally any genetic mutation. Together with a repair template, a correction of the defective locus becomes possible. This book offers insight into the modern tools of genome editing, their hurdles and their huge potential. A new era of in vivo genetic engineering has begun

Inborn errors of metabolism: Lessons from iPSC models

The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs

Perks and considerations when targeting functional non-coding regions with CRISPR/Cas9

Since the CRISPR system was discovered as an adaptive immune response in prokaryotic cells, the past decade has witnessed the engineering and deployment of CRISPR/Cas9 as one of the most efficient and powerful molecular tools. By leveraging the nuclease activity of CRISPR/Cas9, researchers are able to probe the biological functions of genetic elements and dissect molecular interactions by disrupting, activating or inactivating genes. In addition to biological research, the CRISPR/Cas9 toolkit has profoundly revolutionized gene therapy and agricultural products. However, there are many challenges regarding its efficiency, specificity and safety. Continuous efforts are being made to advance techniques and characterize the consequences of genome editing. In this thesis, we describe considerations when targeting genomic regions with CRISPR/Cas9 and provide methods to address some concerns related to efficiency and safety. In Paper I, we introduced a non-hazardous method of transfecting human cells with large-size CRISPR/Cas9 vectors. By co-transfecting small-size vectors (3 kb) to cells, the delivery efficiency of CRISPR/Cas9 vectors (15 kb) and cell viability was significantly increased. The performance of the method has been verified in a number of hard-to-transfect human cell lines with both electroporation- and liposome-based transfection. In Paper II, we revealed the complexity of CRISPR/Cas9-induced on-target genomic alterations by combining an advanced droplet-based target enrichment method followed by long-read sequencing and de novo assembly-based analysis. This approach enabled us to dissect the on-target sequence content in the order of kilobases, which was very challenging with many other available methods. With this tool, we uncovered the co-occurrence of multiple on-target rearrangements including duplication, inversion, as well as integrations of exogenous DNA and clustered interchromosomal rearrangements in CRISPR/Cas9-modified human cells. Furthermore, our study demonstrated that unintended genomic alterations could lead to the expression of DNA derived from both the target region and exogenous sources, as well as affect cell proliferation. In Paper III, we reported a large unexpected genomic deletion in the HAP1 cell line, which is the one of most popular models used in CRISPR/Cas9-mediated experiments. This 287 kb deletion located on Chromosome 10 contains four widely-expressed protein-coding genes including the PTEN gene locus. We detected changes in histone acetylation and transcriptomes in HAP1 cells carrying the deletion. The loss of this genomic locus was not induced by Cas9 off-target nuclease activity. However, the generation of CRISPR/Cas9-modified cells significantly enhanced the frequency of the deletion among cell clones. Furthermore, our analysis indicated that this deletion initially found in HAP1 cells resembled a frequent deletion pattern driven by the PTEN gene in cancer patients. In conclusion, we have presented two methods: one to improve delivery efficiency and another to detect on-target sequence content with higher resolution. Furthermore, we have revealed unintended genomic aberrations at targeted and non-targeted sites. These observations should be taken into consideration when modifying the genome with CRISPR/Cas9, and a comprehensive genomic validation is necessary

Area-wide Integrated Pest Management

Over 98% of sprayed insecticides and 95% of herbicides reach a destination other than their target species, including non-target species, air, water and soil. The extensive reliance on insecticide use reduces biodiversity, contributes to pollinator decline, destroys habitat, and threatens endangered species. This book offers a more effective application of the Integrated Pest Management (IPM) approach, on an area-wide (AW) or population-wide (AW-IPM) basis, which aims at the management of the total population of a pest, involving a coordinated effort over often larger areas. For major livestock pests, vectors of human diseases and pests of high-value crops with low pest tolerance, there are compelling economic reasons for participating in AW-IPM. This new textbook attempts to address various fundamental components of AW-IPM, e.g. the importance of relevant problem-solving research, the need for planning and essential baseline data collection, the significance of integrating adequate tools for appropriate control strategies, and the value of pilot trials, etc. With chapters authored by 184 experts from more than 31 countries, the book includes many technical advances in the areas of genetics, molecular biology, microbiology, resistance management, and social sciences that facilitate the planning and implementing of area-wide strategies. The book is essential reading for the academic and applied research community as well as national and regional government plant and human/animal health authorities with responsibility for protecting plant and human/animal health

Development of a CRISPR-based gene therapy approach to correct duplications causing Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy is a severe neurodegenerative disorder caused by deletions, duplications or point mutations in the DMD gene, which encodes dystrophin. In absence of dystrophin, muscle fibres degenerate and patients become wheelchair dependent by their early teens. Cardiac and respiratory muscles are also affected, causing premature death by the third decade of life. Among the approaches currently being tested in clinical trials to treat this disease, none is suitable to permanently restore dystrophin by removing either small or large multi-exon dystrophin duplications, which account for 10-15% of DMD cases. In this thesis, I designed a genome editing approach to correct duplications in the DMD gene by using a single CRISPR/Cas9 target site. First, I identified a CRISPR/Cas9 nuclease able to efficiently target DMD intron 9, which would be suitable for gene editing in patients harbouring DMD duplications in the mutational hotspot 2-201. Then, I tested both integrating lentiviral particles and nuclear electroporation as tools to deliver and express CRISPR/Cas9 in patient-derived cells carrying different dystrophin duplications. Patient-derived myoblasts allowed me to assess dystrophin restoration at the genomic, transcriptional and protein level by means of the T7 assay, quantitative-PCR and western blot, respectively. I confirmed dystrophin correction in transduced as well as electroporated cells expressing CRISPR/Cas9, and I demonstrated that both a constitutive and a transient nuclease expression led to a similar extent of protein restoration of around 50%. These outcomes allowed me to conclude that CRISPR/Cas9 editing tool is a suitable approach to remove large genomic duplications in vitro. Furthermore, the data presented in this thesis provides the basis for the design of new therapeutic approaches to be tested in vivo in Duchenne Muscular Dystrophy animal models. These include both in vivo CRISPR/Cas9-mediated gene therapy and cell-therapy based on transplantation of ex vivo corrected myoblasts expressing corrected wild-type dystrophin

Development of Chimeric Cas9 Nucleases for Accurate and Flexible Genome Editing

There has been tremendous amount of effort focused on the development and improvement of genome editing applications over the decades. Particularly, the development of programmable nucleases has revolutionized genome editing with regards to their improvements in mutagenesis efficacy and targeting feasibility. Programmable nucleases are competent for a variety of genome editing applications. There is growing interest in employing the programmable nucleases in therapeutic genome editing applications, such as correcting mutations in genetic disorders. Type II CRISPR-Cas9 bacterial adaptive immunity systems have recently been engineered as RNA-guided programmable nucleases. Native CRISPR-Cas9 nucleases have two stages of sequence-specific target DNA recognition prior to cleavage: the intrinsic binding of the Cas9 nuclease to a short DNA element (the PAM) followed by testing target site complementarity with the programmable guide RNA. The ease of reprogramming CRISPR-Cas9 nucleases for new target sequences makes them favorable genome editing platform for many applications including gene therapy. However, wild-type Cas9 nucleases have limitations: (i) The PAM element requirement restricts the targeting range of Cas9; (ii) despite the presence of two stages of target recognition, wild-type Cas9 can cleave DNA at unintended sites, which is not desired for therapeutic purposes; and (iii) there is a lack of control over the mutagenic editing product that is procuded. In this study, we developed and characterized chimeric Cas9 platforms to provide solutions to these limitations. In these platforms, the DNA-binding affinity of Cas9 protein from S. pyogenes is attenuated such that the target site binding is dependent on a fused programmable DNA-targeting-unit that recognizes a neighboring DNA-sequence. This modification extends the range of usable PAM elements and substantially improves the targeting specify of wild type Cas9. Furthermore, one of the featured chimeric Cas9 variants developed in this study has both robust nuclease activity and ability to generate predictable uniform editing products. These superior properties of the chimeric Cas9 platforms make them favorable for various genome editing applications and bring programmable nucleases one step closer to therapeutic applications

Synthesis of new contrast agents for biomedical applications

Several different metal complexes are currently used in diagnostics and therapy (MRI, PET, SPECT, OI, TAT etc.). For their preparation a chelating agent (CA) is mandatory to hold the metal ion, to reduce its toxicity and to target its activity. Polyaminocarboxylates and polyaminophosphonates represent the best choice of CAs for the preparation of metal complexes exploited as imaging probes. These scaffolds ensure high thermodynamic and kinetic stability of the corresponding metal complex. However, their preparation is not always straightforward. During this PhD the research work was focused on the following three main objectives: 1) A strategy for the 1,7-heterodiprotection of cyclen with orthogonal protecting groups have been realised. This protection method is based on the control of pH to implant two different and orthogonal protecting groups in the cyclen scaffold. 2) This protection method has been exploited to prepare two novel bifunctional chelating agents (BFCAs) bearing a phosphonic functionality (DO2AP(tBu)4 and DO3AP(tBu)4). These two novel BFCAs have been designed to prepare innovative and stable metal complexes conjugated with vectors such as antibodies, peptides, nucleotides etc. 3) Three innovative CAs have been prepared and characterised: - TRASUTA is a hexadentate mesocyclic chelating agent and a rigidified AAZTA-like ligand for the coordination of Ga(III). - Cb-tebpm is a cyclam-based chelating agent, designed to prepare stable complexes with gadolinium or in general with lanthanides. - HB-DO3A is an HP-DO3A homologue, optimised to be a strong Gd(III) chelator. HBDO3A has been investigated in its safety profile. In vitro data have shown that, if compared to clinically approved MRI CAs, HB-DO3A presents low affinity to collagen. This characteristic could confer HB-DO3A the advantage of a faster and complete clearance after administration to the patient, with less release of toxic Gd(III) and less side effects

Mitochondria: From Physiology to Pathology

Mitochondria play an increasingly central role in the context of cellular physiology. These organelles possess their own genome (mtDNA), which is functionally coordinated with the nuclear genome. Mitochondrial gene expression is mediated by molecular processes (replication, transcription, translation, and assembly of respiratory chain complexes) that all take place within the mitochondria. Several aspects of mtDNA expression have already been well characterized, but many more either are under debate or have yet to be discovered. Understanding the molecular processes occurring in mitochondria also has clinical relevance. Dysfunctions affecting these important metabolic ‘hubs’ are associated with a whole range of severe disorders, known as mitochondrial diseases. In recent years, significant progress has been made to understand the pathogenic mechanisms underlying mitochondrial dysfunction; however, to date, mitochondrial diseases are complex genetic disorders without any effective therapy. Current therapeutic strategies and clinical trials are aimed at mitigating clinical manifestations and slowing the disease progression to improve the quality of life of patients. The goal of the Special Issue ‘Mitochondria: from Physiology to Pathology’ published in Life (ISSN: 2075-1729) was to collect research and review articles covering the physiological and pathological aspects related to mtDNA maintenance and gene expression, mitochondrial biogenesis, protein import, organelle metabolism, and quality control