建立番茄與番椒以單一原生質體快速評估CRISPR/Cas9核糖核蛋白基因編輯效率平台
Other Title
Establishment of a Single-Protoplast-Based Platform for Evaluating the Efficiency of CRISPR/Cas9 Ribonucleoprotein-Mediated Gene Editing in Tomato and Pepper
Journal
台灣農業研究
Journal Volume
75
Journal Issue
3
Start Page
509
End Page
520
ISSN
2790-086X
Date Issued
2026-09-11
Author(s)
Abstract
茄科中有許多全球重要之營養與糧食作物，為加速茄科作物之品種改良，本研究旨在建立一套利用單一原生質體進行無外源 DNA (DNA-free) 基因編輯體系之效率快速評估平台。以番茄「台南亞蔬 6 號」(‘Tainan AVRDC No. 6’) 與番椒「橙辣」(‘Cheng-la’) 比較不同重量無菌瓶苗葉片在相同酵素條件處理下之原生質體分離效率，並針對感病相關的番茄 SlDMR6 基因與番椒 CaMlo2 基因設計成對嚮導 RNA (dual gRNA)，原生質體經由聚乙二醇 (polyethylene glycol; PEG) 轉殖法直接導入 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 核糖核蛋白 (ribonucleoprotein; RNP)，接續以單一原生質體為單位利用聚合酶鏈鎖反應 (polymerase chain reaction; PCR) 分析平台評估 CRISPR/Cas9 RNP 基因編輯之效率。結果顯示目標基因片段缺失達成效率，在番茄與番椒之原生質體中分別為 33.3% 與 19.2%。進一步以桑格定序確認番茄目標序列精準地產生 154 bp (base pair) 缺失；而番椒目標序列定序結果則呈現多樣性，分別有片段缺失、鹼基置換等多種編輯形式。本研究顯示利用單一原生質體為評估單位之基因編輯快速評估平台，可大幅縮短基因編輯前期對 CRISPR/Cas9 RNP 成效評估所需之時間，為新興育種技術實施提供更快速、精準之評估方法。
Solanaceous species include many globally significant nutritional and food crops. To accelerate variety improvement, this study aimed to establish a rapid platform for evaluating the efficiency of a DNA-free gene-editing system using a single-protoplast-based approach. Using the tomato cultivar ‘Tainan-AVRDC No. 6’ and the chili pepper cultivar ‘Cheng-la’ as experimental materials, we first compared the protoplast isolation efficiency from axenic seedling leaves of different weights under consistent enzymatic conditions. Dual guide RNAs (dual gRNAs) were designed to target the susceptibility genes SlDMR6 in tomato and CaMlo2 in pepper. CRISPR/Cas9 ribonucleoproteins (RNPs) were directly delivered into protoplasts via PEG-mediated transformation. Subsequently, the gene-editing efficiency of the CRISPR/Cas9 RNPs was evaluated at the single-protoplast level using a PCR-based analysis platform. The results indicated that the efficiency of target fragment deletions was 33.3% in tomato protoplasts and 19.2% in pepper protoplasts. Further validation via Sanger sequencing confirmed a 154-bp (base pair) deletion in the tomato target gene. In contrast, sequencing results for the pepper target gene showed diversity, including various editing patterns such as fragment deletions and base substitutions. This study demonstrates that a rapid evaluation system using a single-protoplast-based platform can significantly shorten the time required for preliminary efficacy testing of CRISPR/Cas9 RNPs, providing a faster and more precise evaluation method for the implementation of New Breeding Techniques (NBTs).
Subjects
原生質體
基因編輯
CRISPR/Cas9 核糖核蛋白
成對嚮導 RNA
Protoplast
Gene editing
CRISPR/Cas9 RNP
Dual gRNA
Publisher
農業部農業試驗所
Type
journal article
File(s)![Thumbnail Image]()
Loading...
Name
75-3-11.pdf
Size
3.03 MB
Format
Adobe PDF
Checksum
(MD5):57c58d9727f26670d7531da775e9d927