CRISPR/Cas9-mediated efficient targeted mutagenesis in grape in the first generation

Area of Science:

• Plant genomics
• Molecular biology
• Agricultural science

Background:

• The CRISPR/Cas9 system is a powerful tool for plant genome editing.
• Previous studies demonstrated CRISPR/Cas9 efficiency in grape suspension cells, but biallelic mutations in the first generation remained unconfirmed.

Purpose of the Study:

• To establish the efficacy of the CRISPR/Cas9 system for generating biallelic mutations in the first generation of grape (Vitis vinifera).
• To investigate the potential of CRISPR/Cas9 for gene functional analysis and molecular breeding in grape.

Main Methods:

• Designed four guide RNAs targeting the VvWRKY52 transcription factor gene.
• Utilized Agrobacterium-mediated transformation of Thompson Seedless grape somatic embryos.
• Analyzed first-generation transgenic plants for mutations and off-target effects.

Main Results:

• Achieved 22 mutant plants out of 72 T-DNA-inserted plants.
• 15 lines exhibited biallelic mutations, and seven were heterozygous.
• Observed various editing events, including large deletions; no off-target mutations were detected.
• Knockout of VvWRKY52 conferred increased resistance to Botrytis cinerea.

Conclusions:

• The CRISPR/Cas9 system enables precise genome editing in the first generation of grape.
• This system is valuable for Vitis vinifera gene functional analysis and molecular breeding.
• CRISPR/Cas9 application in grape can lead to enhanced disease resistance through targeted gene modification.