CRISPR/Cas9‐mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV‐U1 strain in regenerated Nicotiana tabacum

Summary

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.