Harnessing CRISPR/Cas9 for Enhanced Disease Resistance in Hot Peppers: A Comparative Study on -Gene-Editing Efficiency across Six Cultivars.

The Mildew Locus O () 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 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/sgRNA2-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, sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/sgRNA1-RNP system to achieve efficient gene editing at the locus in different cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with -mediated enhanced disease resistance.