Efficient genome editing of wild strawberry genes, vector development and validation

Area of Science:

• Plant biotechnology
• Genomics
• Horticultural science

Background:

• The CRISPR-Cas9 system offers powerful genome editing capabilities but has limited reported applications in horticultural plants, particularly concerning germline transmission of mutations.
• Efficient genome editing tools are crucial for improving crop traits in economically important horticultural species.

Purpose of the Study:

• To develop and validate a high-efficiency CRISPR-Cas9 system for genome editing in the wild strawberry (Fragaria vesca).
• To investigate the mutation of auxin biosynthesis and response genes (TAA1 and ARF8) using this system.
• To assess the germline transmission efficiency of induced mutations in subsequent generations.

Main Methods:

• Utilized CRISPR-Cas9 technology with both Arabidopsis (AtU6-26) and wild strawberry (FveU6-2) U6 promoters to drive sgRNA expression for targeted gene editing.
• Applied the system to mutate the TAA1 and ARF8 genes in Fragaria vesca.
• Analyzed the T0 transgenic plants and their progeny (T1 generation) to evaluate mutation efficiency and germline transmission, including large deletions.

Main Results:

• Achieved high-efficiency genome editing in wild strawberry using both tested U6 promoters.
• Successfully induced mutations in the TAA1 and ARF8 genes, with new mutations, including large deletions, detected at high efficiency in the T1 progeny.
• Identified that T1 plants with homozygous arf8 knockout mutations exhibited significantly faster growth compared to wild-type plants.

Conclusions:

• The developed CRISPR vectors are effective for high-efficiency genome editing in the wild strawberry.
• Both sgRNA design and the choice of U6 promoter are critical factors for successful genome editing in this species.
• This research provides a robust platform for genetic engineering of strawberry and other horticultural crops to enhance desirable traits.