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Studies on identification and evaluation of CRISPR diversity on human skin microbiome for development of a new personal identification method

豊間根, 耕地 北海道大学

2022.12.26

概要

近年のDNA配列解析技術の⾰新に伴ってマイクロバイオーム研究が急速に進展し、ヒトの体表を含めた環境中には、環境毎にその系統や組成が異なった微⽣物の集団が⽣存していることが明らかにされてきている。法科学領域においてはマイクロバイオームの構造が環境によって異なることを応⽤して、ヒト⽪膚マイクロバイオーム解析による個⼈識別法の開発を⽬指す研究が各国で進められている。近年のマイクロバイオーム研究は⼤きく⼆つの⼿法によって実施されており、⼀つがアンプリコンシーケンス解析、他⽅がショットガンメタゲノム解析である。従来⽤いられることが多かったアンプリコンシーケンス解析は、PCRにより16SrRNA配列などのマーカー遺伝⼦を増幅し、網羅的に解読する⼿法である。しかしながら16SrRNA配列は近縁種では類似しており、しばしば属単位までの系統情報しか得られないことがある。そのため、個⼈識別においては解像度が低いと⾔われてきた。他⽅、ショットガンメタゲノム解析は試料中のDNAを断⽚化し、これを網羅的に解読する⼿法で、⾼い解像度でマイクロバイオームの構造を把握することができる。しかし、解析にかかるコストが⾼く、PCRによる増幅を経ないために多量のDNAが必要となるといった難点を抱える。このような背景から、ヒト⽪膚マイクロバイオーム解析による個⼈識別法の実現には、16SrRNA配列よりも⾼い解像度で微⽣物の群集構造を捉えるマーカー遺伝⼦を利⽤した、新規アンプリコンシーケンス解析法を開発する必要があると考えられる。

CRISPRは原核⽣物の持つリピート配列で、Casタンパク質と共に原核⽣物における「獲得免疫系」を担う。CRISPRはその機能を果たす上で、細胞内へ侵⼊してきたファージを中⼼とする可動性遺伝因⼦のDNA配列を、リピート配列中にスペーサーと呼ばれる配列として取り込む性質を持つ。このような性質からCRISPR配列の多様性は環境中のウイルス叢の構造を反映している。ヒトの⽪膚におけるウイルス叢は細菌叢に⽐べて個⼈間の多様性が⼤きいことが知られており、本研究ではCRISPRをマーカーとする新規アンプリコンシーケンス解析法を開発し、CRISPRの多様性に基づいた個⼈識別の法科学領域における有⽤性を検討することを⽬的とした。

第I章では、公開データベースに登録されたデータセットを利⽤して、⽪膚マイクロバイオーム中に存在するCRISPRを再構築し、複数⼈の間で共有されるCRISPRのリピート配列を同定した。また、再構築されたCRISPRについて、その配列をリファレンスとなるCRISPRと⽐較したところ、再構築されたCRISPRのスペーサー配列はいずれもリファレンスCRISPRには存在しない独⾃の配列であり、マーカーとしての有⽤性が⽰唆された。

第II章では、3種類の候補CRISPRについて、スペーサー配列をアンプリコンシーケンスにより解析した。性能評価のために16SrRNA配列と⽐較したところ、スペーサー配列の多様性は16SrRNA配列の多様性より個⼈間の多様性が⼤きく、⾼い正答率で個⼈を識別可能であった。このことはCRISPRの配列解析が16SrRNAの配列解析に⽐べ、個⼈識別⽤途に優れる可能性を⽰唆するものである。

第III章では、解析系のDNA抽出法を最適化するために、第II章で使⽤した細菌叢解析に使⽤されるDNA抽出法と、⾃動抽出装置を⽤いた法科学領域で使⽤されるDNA抽出法の、2種類のDNA抽出法を⽐較した。16SrRNA配列のコピー数の定量とCRISPRの多様性評価の結果、2種類のDNA抽出法は細菌DNAの抽出効率について同等であり、CRISPRの多様性はDNA抽出法によらず安定していると考えられた。また、試料の識別精度も同等であり、法科学領域で使⽤される⾃動抽出装置によって抽出されたDNAがCRISPRの多様性評価にも使⽤可能であることが⽰された。

メタゲノムデータセットから同定されたCRISPRのアンプリコンシーケンス解析により、⽪膚上の連鎖球菌に由来するCRISPRの多様性が個⼈特異的であることが明らかになった。本研究は概念実証ではあるが、開発したCRISPR解析法は従来のDNA型検査法を補完する⼿法として、法科学領域において応⽤可能であると考えられる。今後もさらなる解析系の最適化と法科学的評価進めることで、実⽤化に向けた課題の解決を⽬指し、さらなる解析系の最適化と法科学的評価が必要である。

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