應用先導編輯修飾水稻EPSPS基因

Abstract

基因編輯技術可以精準地在目標 DNA產生期望之插入(insertion)、刪除(deletion)以及取代(substitution)突變 ，而CRISPR/Cas9為當今基因編輯最有潛力的工具，但先前研究多僅利用Cas9蛋白產生雙股DNA斷裂，進而選擇修復錯誤之個體並觀察基因剔除(knock-out)產生的結果。最近發展的先導編輯(prime editing)技術可利用連接反轉錄酶的Cas9融合蛋白直接於指定位置創造期望突變。 本研究利用先導編輯使水稻內生EPSPS基因產生新功能(gain-of-function)，透過取代其中兩個胺基酸使水稻由對嘉磷塞敏感型轉為抵抗型，兩點的突變分別為第172個glycine突變為alanine及第215個glycine為aspartic acid。透過農桿菌轉殖法，於台農67號水稻轉入辨識突變位置的pegRNA、兩位點各自的nicking spacer及nCas9-RT基因。最終成功獲得6株轉殖系，其中3株更在其中G215突變位置成功偵測到期望突變的發生 。

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 modifications, while most efforts results in “knock-out” of a gene because of DNA double strands break by Cas9. Recently, “Prime editing” 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, PE (prime editing) technique was used to create a “gain-of-function mutant” EPSPS gene of rice for glyphosate resistant by replacing two amino acids. The replacing indicates the codon for the glycine residue at position 172 was changed to encode an alanine residue and the codon for the glycine at position 215 was changed to encode an aspartic acid residue. The specific pegRNA as well as nicking RNA constructs were synthesized, together with nCas9-RT gene, and transformed into rice TNG67. Successful transgenic rice plants were confirmed and three of them have been detected expected point mutation.