This detailed volume provides a comprehensive collection of protocols for epigenomic research, powering our ability to analyze epigenetic modifications across the entire genome. Beginning with methods used to investigate epigenomic modifications such as DNA methylation, histone modifications, and chromatin structure, the book continues with methods for manipulating the epigenome, including platforms for epigenome editing, inducible systems for epigenome editing, and epigenetically modified animals. Written for the highly successful Methods in Molecular Biology series, chapters feature introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Epigenomics: Methods and Protocols serves as an ideal resource for researchers looking to further expand the utility and scope of epigenomics research.

Heritable human genome editing - making changes to the genetic material of eggs, sperm, or any cells that lead to their development, including the cells of early embryos, and establishing a pregnancy - raises not only scientific and medical considerations but also a host of ethical, moral, and societal issues. Human embryos whose genomes have been edited should not be used to create a pregnancy until it is established that precise genomic changes can be made reliably and without introducing undesired changes - criteria that have not yet been met, says Heritable Human Genome Editing. From an international commission of the U.S. National Academy of Medicine, U.S. National Academy of Sciences, and the U.K.'s Royal Society, the report considers potential benefits, harms, and uncertainties associated with genome editing technologies and defines a translational pathway from rigorous preclinical research to initial clinical uses, should a country decide to permit such uses. The report specifies stringent preclinical and clinical requirements for establishing safety and efficacy, and for undertaking long-term monitoring of outcomes. Extensive national and international dialogue is needed before any country decides whether to permit clinical use of this technology, according to the report, which identifies essential elements of national and international scientific governance and oversight.

Over the past decades, chromatin remodelling has emerged as an important regulator of gene expression and plant defense. This book provides a detailed understanding of the epigenetic mechanisms involved in plants of agronomic importance. The information presented here is significant because it is expected to provide the knowledge needed to develop in the future treatments to manipulate and selectively activate/inhibit proteins and metabolic pathways to counter pathogens, to treat important diseases and to increase crop productivity. New approaches of this kind and the development of new technologies will certainly increase our knowledge of currently known post-translational modifications and facilitate the understanding of their roles in, for example, host-pathogen interactions and crop productivity. Furthermore, we provide important insight on how the plant epigenome changes in response to developmental or environmental stimuli, how chromatin modifications are established and maintained, to which degree they are used throughout the genome, and how chromatin modifications influence each another.

The CRISPR-Cas9 genome-editing system is creating a revolution in the science world. In the laboratory, CRISPR-Cas9 can efficiently be used to target specific genes, correct mutations and regulate gene expression of a wide array of cells and organisms, including human cells. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases is a unique reading material for college students, academicians, and other health professionals interested in learning about the broad range of applications of CRISPR/Cas9 genetic scissors. Some topics included in this book are: the role of the CRISPR/Cas9 system in neuroscience, gene therapy, epigenome editing, genome mapping, cancer, virus infection control strategies, regulatory challenges and bioethical considerations.

Plants are vulnerable to pathogens including fungi, bacteria, and viruses, which cause critical problems and deficits. Crop protection by plant breeding delivers a promising solution with no obvious effect on human health or the local ecosystem. Crop improvement has been the most powerful approach for producing unique crop cultivars since domestication occurred, making possible the main innovations in feeding the globe and community development. Genome editing is one of the genetic devices that can be implemented, and disease resistance is frequently cited as the most encouraging application of CRISPR/Cas9 technology in agriculture. Nanobiotechnology has harnessed the power of genome editing to develop agricultural crops. Nanosized DNA or RNA nanotechnology approaches could contribute to raising the stability and performance of CRISPR guide RNAs. This book brings together the latest research in these areas. CRISPR and RNAi Systems: Nanobiotechnology Approaches to Plant Breeding and Protection presents a complete understanding of the RNAi and CRISPR/Cas9 techniques for controlling mycotoxins, fighting plant nematodes, and detecting plant pathogens. CRISPR/Cas genome editing enables efficient targeted modification in most crops, thus promising to accelerate crop improvement. CRISPR/Cas9 can be used for management of plant insects, and various plant pathogens. The book is an important reference source for both plant scientists and environmental scientists who want to understand how nano biotechnologically based approaches are being used to create more efficient plant protection and plant breeding systems. Shows how nanotechnology is being used as the basis for new solutions for more efficient plant breeding and plant protection Outlines the major techniques and applications of both CRISPR and RNAi technologies Assesses the major challenges of escalating these technologies on a mass scale

Although all cell types in a multicellular organism contain the same genetic information, they are functionally and morphologically different. Such diversity can only be achieved by a highly regulated spatial and temporal control of gene expression by over 2000 transcription factors and chromatin regulators. The utilization of these transcriptional effector domains in gene regulation tools, as in CRISPRi/a and genetic circuits, have been instrumental in interrogating regulatory elements and gene function and holds promise for therapeutic gene or cell therapy applications. However, there is a need for new tools that do not rely on overexpressing effector domains and are small enough to deliver and combine for use in gene regulation tools. There is also the lack of systematic understanding of how effector domains regulate transcription across genomic and cell-type contexts and how their function depends on cellular cofactors. Characterizing the context of how these effectors function will help in the development of gene regulation tools that can perform across different contexts. First (in chapter 2), I develop a strategy for gene control using small single domain antibodies, called nanobodies, that bind and recruit endogenous chromatin regulators to a gene. I show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against two different types of repressive chromatin regulators, HP1 and DNMT1, can silence a fluorescent reporter gene. Combining these nanobodies together or with other regulators, such as DNMT3A or KRAB, I observe enhanced silencing speed and epigenetic memory. Finally, I use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using compact nanobodies against chromatin regulators for controlling gene expression and epigenetic memory. To better understand how the dynamics and mechanisms of chromatin-mediated gene control can vary in different cell types (chapter 3), I study the effect of two repressive histone methyltransferases, KRAB and EED, on gene expression and epigenetic memory at a fluorescent reporter gene in mouse embryonic stem cells (mESCs) and Chinese hamster ovary (CHO-K1) cells. I find that, when compared to CHO-K1 cells, KRAB had slower rates of silencing and that EED can only partially silence in mESCs. Moreover, silencing mediated by these effectors had shorter duration of epigenetic memory in mESCs, such that EED silencing was completely reversible. Overall, these results provide a preliminary look into the cell-type specific function of these regulators in stem cells and acts as a steppingstone for future experiments characterizing more transcriptional effectors across different contexts, such as cell-type, gene target, and DNA-binding domain. Lastly (in chapter 4), I used high-throughput recruitment (HT-recruit) to measure effector function for a pooled library of about 6,000 nuclear protein domains across 15 target gene, cell-type, and DNA-binding domain contexts. I discover context-dependent effectors (e.g., WW and HLH domains) and identify other context-independent effectors that can improve transcriptional perturbation tools (e.g., the tripartite activator NFZ and the ZNF705F KRAB repressor). Moreover, using CRISPR screens, we identify genetic dependencies for the context-dependent HLH repressors. Ultimately, these results reveal the context-dependence across human effectors, demonstrates how rare cell type- and target-specific effectors can be systematically characterized by combined high-throughput protein domain and genetic perturbation screens, and enables a new suite of transcriptional control tools for research and therapeutic applications. Collectively, this dissertation demonstrates my work in discovering and engineering epigenetic tools that allow for better and more potent transcriptional control across different genomic and cell-type contexts, in hopes of improving upon the existing gene regulation toolbox.