embedded as a routine technique in molecular and cell biology laboratories across the field. New industries have been created to supply CRISPR reagents and CRISPR‐edited cell and animal models to the research scientist, to develop CRISPR medicines and to create CRISPR diagnostics. The applications and impact of CRISPR in drug discovery are discussed at length within this book. Within eight short years, CRISPR has transformed our ability to identify and characterize the role of new drug targets in disease and to create the cell and animal models integral to identify and optimize drug candidates. With the rate of innovation in this field, we can look forward to the development of novel CRISPR systems that increase the efficiency and specificity of gene editing, to the development of transformative CRISPR therapies with the potential to cure severe genetic diseases and to the invention of highly sensitive diagnostics for the early identification and subsequent cure of many common diseases. As we move through the coming decades, the opportunity for CRISPR to improve human health remains enormous.
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2 Historical Overview of Genome Editing from Bacteria to Higher Eukaryotes
Marcello Maresca
Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
2.1 Introduction
Molecular cloning methods have been instrumental for the establishment of the biotechnological industry. The ability to clone any DNA sequence of interest into a DNA vector has been a key technology advancement toward the generation of cellular and animal model of disease and the development of biopharmaceuticals. Traditional molecular cloning methods mostly rely on restriction enzymes‐mediated digestion and ligation of the digested fragments. Classical restriction enzymes recognize a relatively short DNA sequence and as a consequence, they are too unspecific to be used directly for DNA engineering applications in cellula.
Novel improvements in DNA assembly methods combined with the cost reduction and with the increase in accuracy of DNA synthesis processes have led to the possibility of assembling large DNA constructs in vitro. Synthetic genomes will have a key role in future DNA engineering platforms but they will not be discussed in this chapter and in this book, where we will focus on in cellula genome engineering approaches.
In this chapter, I will give a brief description of the advancements in the precise genome editing field starting from observations of single‐stranded oligonucleotides‐mediated repair in yeasts to Recombineering and CRISPR‐Cas9‐dependent editing. These technologies have all greatly expanded the tools and methods that are used to generate disease models and to develop assays for drug discovery (Figure 2.1).
2.2 Bacterial DNA Engineering (Recombineering)
Microbes and microbial‐derived systems have been extensively used for the development of novel DNA engineering tools and for the application of these tools to DNA cloning. Restriction enzymes, recombinase systems such as CRE/Lox, integrases such as ΦC31‐Int, and the Cas9‐CRISPR system have all microbial origin. Recombinases and integrases‐based systems have been extensively used to engineer the mammalian genomes but we will not discuss them in this book that is focusing on scarless genome engineering systems. This chapter will focus on the development of Recombineering for bacterial engineering and its use in genome engineering with particular focus on applications in drug discovery.
The inspiration for the Recombineering (recombination‐mediated