CRISPR-Cas9技術應用於粒線體基因編輯之優化設計

Abstract

粒線體對於維持細胞內的能量及動態恆定扮演極重要的角色，當粒線體基因受損時會造成粒線體功能部分失活，甚至導致嚴重的遺傳性疾病，例如萊氏症(Leigh syndrome)。目前雖然有許多團隊嘗試利用限制酶或人工合成的DNA內切酶，例如：ZFN或TALEN，來針對突變的粒線體基因進行剪切剔除，但這些方法往往會受到選擇位點不足、製作不易等缺點所限制。近年來迅速發展的基因編輯技術CRISPR-Cas9便可解決上述兩項問題，目前除了三篇仍具有爭議的研究發表外，尚未有其他人提出相關研究成果或重複其研究。因此，在此篇研究中，我們分別在Cas9蛋白及嚮導RNA (guide RNA)上進行一系列不同粒線體標的序列(mitochondria-targeting sequence, MTS)的修飾。根據結果，我們發現帶有MTHFD1L MTS之mito-Cas9蛋白與僅帶有5’端10個核苷酸延長序列之嚮導RNA 51號，這兩者可以最有效率地進入粒線體。我們的結果可望運用CRISPR-Cas9技術來建立一套更加完善的粒線體基因編輯工具，未來將可應用於生物學研究與罕見粒線體疾病之治療。

The mitochondrial genome is responsible for the maintenance of the cellular energy source and homeostasis. Therefore, partial loss of mitochondrial functionality and even devastating diseases happen when the mitochondrial genome is damaged. Nowadays, several strategies like restriction enzymes, ZFNs and TALENs, have been developed to specifically eliminate mutated mitochondrial DNA. However, all methods aforementioned have their own limitations on either limited target site choice or laborious manufactural process. CRISPR-Cas9 is a soaring new DNA editing tool which has not been widely applied in mitochondrial genome engineering, except for three controversial papers published. Consequently, in this study, we aim to establish a more reliable and tractable platform by fusing both Cas9 protein and guide RNA with various mitochondria-targeting sequences (MTSs). Our data show that the mito-Cas9 with the MTS from mitochondrial monofunctional C1-tetrahydrofolate synthase (MTHFD1L) and the mito-guide RNA 51 with only 10-nucleotide extended form the 5’ end both have the best import efficiency into mitochondria. Our study provides a potential technique to edit mitochondrial genome through CRISPR-Cas9 gene tool and may help generate gene therapy for mitochondrial diseases.