PDF(Pubmed)
Abstract：
The Capsicum annuum Mildew Locus O (CaMLO2) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated CaMLO2 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/CaMLO2single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/CaMLO2sgRNA2-RNP systems by extending their application from the bell pepper \'Dempsey\' and the hot pepper \'CM334\' to six commercial hot pepper cultivars. Across the six cultivars, CaMLO2sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas CaMLO2sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/CaMLO2sgRNA1-RNP system to achieve efficient gene editing at the CaMLO2 locus in different Capsicum annuum cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with CaMLO2-mediated enhanced disease resistance.
摘要：
辣椒霉菌基因座O（CaMLO2）基因对于植物对白粉病等真菌病原体的防御反应至关重要，对温室辣椒作物的重大威胁。基因组编辑的最新进展，特别是使用成簇的规则间隔短回文重复序列(CRISPR)/Cas9,为修饰抗病基因和改善作物特性提供了前所未有的机会.然而,CRISPR技术在辣椒品种中的应用受到限制，再生过程仍然具有挑战性。本研究通过研究在六个商业辣椒品种中使用经过验证的CaMLO2遗传剪刀系统的可行性来解决这些限制。我们评估了先前报道的高效Cas9/CaMLO2单向导RNA（sgRNA）1-核糖核蛋白（RNP）和低效率Cas9/CaMLO2sgRNA2-RNP系统的基因编辑效率，方法是从甜椒“Dempsey”和辣椒“CM334”扩展到六个商业辣椒品种。在六个品种中，CaMLO2sgRNA1的编辑效率为6.3％至17.7％，而CaMLO2sgRNA2没有显示编辑效率，突出sgRNA1的优越功效。这些发现表明，无论其品种基因型如何，利用经过验证的Cas9/CaMLO2sgRNA1-RNP系统在不同辣椒品种的CaMLO2基因座上实现有效的基因编辑的潜力。这项研究为开发具有CaMLO2介导的增强抗病性的改良辣椒品种提供了有效的基因组编辑工具。
