Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

Area of Science:

• Plant biotechnology
• Genome editing
• Crop improvement

Background:

• Conventional gene insertion methods in plants often result in random integration or require selectable markers.
• Targeted gene insertion at specific genomic locations (safe harbors) is desirable for predictable gene expression and trait stability.
• Previous targeted insertion methods were limited by DNA fragment size and efficiency.

Purpose of the Study:

• To develop an optimized CRISPR-Cas9-based system for efficient, marker-free targeted insertion of large DNA fragments into rice.
• To assess the impact of targeted gene insertion on carotenoid biosynthesis, plant morphology, and yield in rice.
• To confirm the absence of off-target mutations in CRISPR-Cas9 edited rice.

Main Methods:

• Utilized an optimized CRISPR-Cas9 system for targeted insertion of a 5.2 kb carotenoid biosynthesis gene cassette into two rice genomic safe harbors.
• Employed whole-genome sequencing to detect off-target mutations.
• Phenotypically evaluated engineered rice plants for carotenoid content, morphology, and yield.

Main Results:

• Successfully achieved marker-free targeted insertion of a large DNA fragment (5.2 kb) into rice genomic safe harbors.
• Engineered rice plants exhibited significantly increased seed carotenoid content.
• No detrimental effects on plant morphology or yield were observed, and whole-genome sequencing confirmed the absence of Cas9 off-target mutations.

Conclusions:

• Demonstrated the efficacy of CRISPR-Cas9 genome editing for marker-free, targeted insertion of large DNA payloads in rice.
• This approach provides a powerful tool for the genetic improvement of rice and other crops by enhancing valuable traits like carotenoid biosynthesis.
• The developed method offers a reliable strategy for introducing beneficial genes into predetermined genomic locations without undesirable side effects.