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CRISPR/Cas9システムを用いた逆遺伝学的アプローチによる精子形成メカニズムの解明

大浦, 聖矢 大阪大学 DOI:10.18910/87999

2022.03.24

概要

新しい個体を作り出すことは生命の根源的活動であり、有性生殖を行う生物においては生殖細胞がこの中心的な役割を担う。特に、精巣は、トランスクリプトームが神経系についで複雑なことに加え、最も発現遺伝子数が多いことも知られており、有性生殖生物にとって精子形成が如何に重要で複雑かを物語っている。また、人口爆発や少子高齢化、不妊症といった多様な人口問題を抱える時代に突入した人類にとって、生殖生物学の基礎的な知見の重要性は計り知れない。一例として、非ホルモン性の経口男性避妊薬の開発は、NIHの戦略目標に設定され、開発が急がれている。また、5年に渡って不妊と診断された夫婦を追跡したWHOの調査によると、不妊の原因のうち31%が女性に、22%が男性に、21%が両者に起因することが報告されるなど(先進諸国のデータを使用;残りの12%は妊娠、14%は原因不明)、生物学的意義のみならず、不妊原因究明という観点からも精子形成・精子機能メカニズムの解明が求められる。

 2021年現在においても、雄性生殖細胞をinvitroで受精可能な精子まで分化させた報告はなく、精子形成や精子の研究には、依然として、個体レベルでの解析が求められる。そこで、著者は、当時、過渡期にあったゲノム編集技術Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Proteins 9 (CRISPR/Cas9)システムを用いて、精巣で特異的に発現する遺伝子に対して、順次、遺伝子欠損(Knock-out; KO)マウスを作製するアプローチにより、精子形成メカニズム解明に迫ることとした。

 著者は、「CRISPR/Cas9システムを用いた逆遺伝学的アプローチによる精子形成メカニズムの解明」という壮大な研究テーマに取り組むにあたり、個体レベルでの高効率な精子表現型解析手法の確立(第一章)、また、より広範な配列を標的とできる改変型Cas9の開発(第二章)という技術面から着手した。さらに、これらの技術を応用しながら、技術を駆使しながら遺伝子改変マウスを作製することで、精子形成に必須な遺伝子を同定してきた(第三章)。中でも、不妊を呈した遺伝子改変マウスの詳細な解析により、CFAP97 domain containing 1(Cfap97d1)遺伝子を精子尾部の基本構造である9本の二重微小管の維持に必須である遺伝子(第四章)、potassium channel tetramerisation domain containing 19 (Kctd19) 遺伝子とzinc finger protein 541 (Zfp541)遺伝子を減数分裂の進行に必須の遺伝子(第五章)として同定した。以上のように、CRISPR/Cas9による高効率なKOマウス作製と交配試験による標的遺伝子の生理的表現型スクリーニングという実験スキームにより、今までにない効率で必須遺伝子を同定してきた。精子形成における必須遺伝子の同定は、不妊の原因究明や避妊薬の開発といった応用面を含め、今後、あらゆる生殖生物学研究の礎となりえる。実際、著者の所属研究室では、共同研究ベースで必須遺伝子に特異的に結合する小分子探索も進めており、経口男性避妊薬の有力な候補も見つかりつつある。多様な人口問題を抱える時代に突入した人類にとって、生殖生物学への理解を深めることは急務といっても過言ではない。著者の研究成果であるKOマウスというバイオマテリアル、ならびに、精子形成に関する基礎的な知見が、様々な人口問題を解決する一助になることを願う。

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