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

Plant Biotechnol J. 2023 Dec;21(12):2641-2653. doi: 10.1111/pbi.14159. Epub 2023 Aug 23.

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.

Keywords: CRISPR/Cas9; TMV resistance; gene knock-in; gene replacement; long fragment insertion; protoplast regeneration.

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Gene Editing / methods
  • Genome, Plant
  • Nicotiana* / genetics
  • Plant Breeding
  • Plants / genetics
  • Protoplasts