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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).