CRISPR/Cas9‐mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV‐U1 strain in regenerated <i>Nicotiana tabacum</i>
Yanli Li, Changjun Huang, Yong Liu, Jianmin Zeng, Haiqin Yu, Zhijun Tong, Xinjie Yuan, Xueyi Sui, Dunhuang Fang, Bingguang Xiao, Shancen Zhao, Yuan Cheng
Abstract
CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.