2017-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/661148摘要：由於大多數微型核酸基因 (MIR gene) 長度太小 (大約200-300-nt)，缺乏 T-DNA 插入之突變株。CRISPRC-Cas9 反向遺傳技術能精準進行基因剔除，產生突變株。然而，至今文獻尚未報導此技術能進行微型核酸基因剔除。微型核酸前體 (microRNA precursor) 的髮夾狀二級結構，可供Dicer-like 1 (DCL1) 辨認並產生微型核酸。在微型核酸前體的非關鍵位置插入或丟失幾個核酸並不會改變其二級結構，也不會影響 DCL1 辨認及產生微型核酸。因此，運用 CRISPRC-Cas9 進行微型核酸基因剔除，必須準確破壞其二級結構或是移除微型核酸基因序列，才能有效抑制微型核酸生成。嚮導核酸 (guide RNA) 之設計為決定 CRISPRC-Cas9 能否精準靶定微型核酸基因位置之關鍵因素。然而，現有的嚮導核酸預測軟體皆針對模式生物設計，無法提供非模式生物或微型核酸基因編輯使用。此外，植物基因轉殖系統所使用之 CRISPRC-Cas9 二位元載體 (binary vector) 進行嚮導核酸序列構築之方便性仍有待改善。因此本計畫擬將設計一套轉錄體 (transcriptome) 輔助型嚮導核酸預測系統，提供非模式生物基因與微型核酸基因剔除時脫靶效應 (off-targeting) 之篩選與分析。同時，擬將 Gateway基因重組技術導入植物之 CRISPRC-Cas9 二位元載體，提升嚮導核酸序列構築方便性。本計畫擬用地錢植物 (Marchantia polymorpha) 具有的單倍體優勢來快速驗證所開發之 CRISPRC-Cas9 系統於微型核酸基因剔除能力。此外，我們也將利用此系統剔除阿拉伯芥 (Arabidopsis thaliana) MIR396a/b 基因，獲得 atmir396a/b 雙突變株以便後續進行其微型核酸分子遺傳研究。我們的最終目標為開發 CRISPR-Cas9 微型核酸基因剔除系統，利用轉錄體進行嚮導核酸設計與脫靶效應預測，擴大非模式生物基因編輯之應用。<br> Abstract: Most microRNA (miRNA) genes (MIR genes) lack suitable T-DNA insertion lines due to the small size of the gene bodies (~200-300-nt in length). The CRISPR-Cas9 reverse genetic technique enables high precision gene-editing for the generation of gene knockout mutants; however, no previous studies have reported on the application of CRISPR-Cas9 to MIR gene knockout. The miRNA precursor (pre-miRNA) possesses a hairpin structure for Dicer-like 1 (DCL1) recognition for the generation of miRNA. The deletion or insertion of a few nucleotides in noncritical positions in pre-miRNA would not necessarily affect the production of miRNA by DCL1. Therefore, CRISPR-Cas9-mediated MIR gene-editing requires that the hairpin structure be destroyed or the miRNA sequence removed in order to block the miRNA biogenesis. The design of guide RNA (gRNA) for CRISPR-Cas9 systems is crucial to the precise targeting of critical positions in pre-miRNA. However, all existing gRNA predictors have been designed for the gene-editing of model organisms, not for non-model organisms or the MIR gene. Furthermore, the application of the binary vector for the CRISPR-Cas9 system in plants requires further improvement. The objective of this study is to design a transcriptome-based gRNA predictor for an off-target screening of the MIR gene and the gene-editing of non-model organisms. We will also construct a Cas9-free/Gateway-based CRISPR-Cas9 binary vector for gene-editing in plants. The haploid dominant life cycle of liverwort Marchantia polymorpha will be used to enable the rapid testing of MIR gene-editing using our developed CRISPR-Cas9 system. We will also apply this system to the generation of MIR396a/b double mutants in diploid Arabidopsis to facilitate studies in molecular genetics. Our ultimate objective is to develop a system capable of editing of the MIR gene, as well as providing gRNA and off-target scanning based on transcriptome profiles in order to extend the application of CRISPR-Cas9 to non-model organisms.CRISPR-Cas9嚮導核酸微型核酸地錢非模式物種CRISPR-Cas9gRNA predictormicroRNAMarchantia polymorphanon-model organisms