Genome Editing and Creating Mutant Strains in Medaka

Introduction

In recent years, various genome editing technologies have been developed, such as the TAL effector nuclease (Transcription ActivatorLike Effector Nuclease) and CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein) systems, which allow DSB (Double-Strand Breaks) to be introduced at any desired sequence in the genome. The two are fundamentally different. TAL effector nuclease is an artificial nuclease of chimeric protein which has a nuclease domain. CRISPR/Cas systems utilize an RNA-guided RGEN (RNA-guided endonuclease). Following those developments, a broad variety of tools based on such technology have been developed, and advancements are being made day by day. One of the most significant contributions of this technology is that has now become possible to perform selective gene targeting (gene disruption) using a wider variety of organisms. Until now this was only possible with limited model organisms such as mice, in which ES cells are present. So far, there have been reports of gene disruption with nematodes, drosophila, silkworms, crickets, sea squirts, frogs, newts, and so on.4) The mechanism by which gene targeting using DSB takes place is shown in Fig. 1. After DSB occurs, cells attempt to perform repairs. There are two main paths that can be followed when performing repairs. With one, known as homology-directed repair, normal sister chromosome is used as a template for performing the repair. In this case, chromosomes are restored to their original state, eliminating any damage. As a result, mutations are not introduced. On the other hand, with non-homologous end joining, chromosomes are joined as a stopgap measure to address the chromosome breakage. At this time, a mutation in the form of insertion or deletion (in/del) of the base is introduced, and genetic disruption occurs.

October 12, 2017 GMT