Targeted Mutagenesis

Mutational analysis in forward genetics is an effective strategy in invertebrate model animals and zebrafish in vertebrates. Forward genetic studies in mammalian systems are significantly limited due to difficulties in isolating mutants. Gene knockout technology using homologous recombinationrecombinationhomologous drastically changed research trends in mice. Reverse genetic approaches for targeted mutagenesis allowed a generation of mutations in any cloned genes. This chapter briefly outlines gene-targetinggene-targeting strategies in mice. We also discuss strategies developed for gene targeting by homologous recombination in Drosophila. An essential step for gene targeting is generating double-stranded DNA breaks. Technologies for site-specific DNA cleavage were developed for various applications in genetic engineering. We will discuss Zinc-Finger NucleaseZinc-Finger Nuclease (ZFNZFN) and TALENTALEN as genetically engineered endonucleaseendonucleases designed to target specific DNA sequences. We shall then introduce the findings of the CRISPR-Cas prokaryotic defense systems against viral infection. The CRISPR-Cas system is used for inducing highly efficient double-stranded breaks. We will discuss critical steps for the guide RNAguide RNA formation and the assembly of an RNA-Cas complex for cleaving specific DNA targets. CRISPR-Cas systems have become versatile tools for gene targeting and genome editinggenome editing in many organisms. We will also discuss the applications of the CRISPR-Cas system for genome-wide mutagenesis in mammalian cells and animals.