CRISPR/Cas9-mediated genome editing in wild-derived mice: generation of tamed wild-derived strains by mutation of the a (nonagouti) gene

Michiko Hirose,Hiroko Tsukamura,Tamio Furuse,Kuniya Abe,Hideki Kaneda,Ayumi Hasegawa,Kimiko Inoue,Shigeharu Wakana,Keiji Mochida,Atsuo Ogura,Arata Honda,Tatsuhiko Goto,Yoshihisa Uenoyama,Shogo Matoba,Ikuko Yamada,Yuki Hatanaka

doi:10.1038/srep42476

Abstract

Wild-derived mice have contributed to experimental mouse genetics by virtue of their genetic diversity, which may help increase the chance of identifying novel modifier genes responsible for specific phenotypes and diseases. However, gene targeting using wild-derived mice has been unsuccessful because of the unavailability of stable embryonic stem cells. Here, we report that CRISPR/Cas9-mediated gene targeting can be applied to the Japanese wild-derived MSM/Ms strain (Mus musculus molossinus). We targeted the nonagouti (a) gene encoding the agouti protein that is localized in hair and the brain. We obtained three homozygous knockout mice as founders, all showing black coat colour. While homozygous knockout offspring were physiologically indistinguishable from wild-type litter-mates, they showed specific domesticated behaviours: hypoactivity in the dark phase and a decline in the avoidance of a human hand. These phenotypes were consistent over subsequent generations. Our findings support the empirical hypothesis that nonagouti is a domestication-linked gene, the loss of which might repress aggressive behaviour.

Highlights

The mouse (Mus musculus) is the most frequently used mammalian species for biomedical research because of its defined genetic background and the relative ease of carrying out genetic modification
In wild-derived strains have been more difficult to achieve in comparison to laboratory strains because they are generally resistant to assisted reproductive technologies (ARTs)
This is compounded by the unavailability of stable embryonic stem cells (ESCs) lines, which have been the main tools for generating targeted genetic modifications

Summary

Introduction

The mouse (Mus musculus) is the most frequently used mammalian species for biomedical research because of its defined genetic background and the relative ease of carrying out genetic modification. The genetic diversity of classical laboratory mouse strains is thought to be limited because of closed breeding using genetically related ancestral strains[2] Such domesticated mice are more prone to many types of tumours and metabolic diseases, such as diabetes, when compared with other species[1]. In wild-derived strains have been more difficult to achieve in comparison to laboratory strains because they are generally resistant to assisted reproductive technologies (ARTs) This is compounded by the unavailability of stable ESC lines, which have been the main tools for generating targeted genetic modifications. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] have already been applied successfully to gene targeting in laboratory mice[10,11] One of their most important advantages is that gene-targeted mice can be generated without the intervening steps of generating ESCs and germ line chimaeras. Phenotypic analysis of the mutant mice provided the first experimental evidence of the nonagouti gene as a domestication-linked gene

Methods

Results

Conclusion