應用先導編輯修飾大豆儲存蛋白基因之初探

Abstract

基因編輯(gene editing)技術應用個別基因專一序列，定點突變目標基因，產生插入(insertion)、刪除(deletion)、置換(substitution)等突變，而CRISPR/Cas9為當今最廣泛被應用的工具。CRISPR/Cas9主要使目標基因產生插入/刪除(indel)突變而失去功能(loss of function)，最近發展的先導編輯(prime editing)技術則利用連接反轉錄酶的Cas9 nickase，能夠在指定位置專一且有效地造成目標基因之指定鹼基對發生置換，使得該基因有機會獲得新功能(gain of function)。本研究旨在利用先導編輯使大豆的儲存蛋白 Glycinin基因之15個修飾位點發生置換，在不改變總蛋白含量以及影響蛋白結構為前提之下提升原本較缺乏的硫胺基酸Methionine(Met)。透過農桿菌轉殖技術，目前已成功將其中A410、I070、V429、L123、L386等5個辨識突變位點的pegRNA與所需之Cas9融合蛋白構築轉入Williams 82大豆，雖然尚未從T0轉植株中找到發生預期突變之個體，後續將繼續確認更多的轉基因大豆並檢測其突變概況，篩選指定編碼修飾為ATG之子代，分析其Met與總蛋白含量。為了往後能夠有效且大量地篩選突變株，本研究先行使用轉基因水稻透過自動移液器與三維振列的結合，成功建立一套能夠有效率地分析轉殖株的篩選系統。

Gene editing techniques can achieve specific and precise gene modification by DNA insertion, deletion or substitution. CRISPR/Cas9 system has been proven as the most powerful tool for genome modification, while most efforts result in knock-out of gene because of DNA double strands break by Cas9. Recently, Prime Editing(PE) was developed that directly writes desired codon into a specified DNA site using a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase. In this study, prime editing was used to edit soybean storage protein gene for encoding more methionine without affecting the total protein content and its structure. By Agrobacterium transformation system, five pegRNAs that recognize the desired mutation sites and required Cas9 fusion protein have been successfully transferred into Williams 82 soybean, however, we haven’t confirmed any desired mutation occurred. We are going to create more successful transgenic soybean plants, harboring desired target (ATG) for protein content, T-DNA free, and off-target analyses. The successful outcome may provide an alternative breeding method of soybean.