A Fast and Cost-Effective Genotyping Method for CRISPR-Cas9-Generated Mutant Rice Lines

Area of Science:

• Plant science
• Molecular biology
• Genetics

Background:

• High-throughput generation and phenotyping of mutant plant lines necessitate efficient genotyping.
• Traditional genotyping workflows involve time-consuming and costly steps like DNA purification and bacterial cloning.

Purpose of the Study:

• To develop and validate an alternative, streamlined genotyping workflow for plant research.
• To compare the efficiency, cost, and quality of the proposed workflow against traditional methods.

Main Methods:

• Utilized Phire polymerase on fresh plant tissue and ExoProStar treatment for sequencing library preparation.
• Generated CRISPR-Cas9 mutants for ZAXINONE SYNTHASE (ZAS) in rice using two guide RNAs.
• Compared results from the proposed workflow with a traditional workflow for genotyping nine T1 plants, employing automated analysis systems.

Main Results:

• The proposed workflow yielded results of comparable quality to the traditional method.
• The new workflow reduced genotyping time from 3 days to 1 day.
• The cost was reduced by approximately 35 times, with fewer steps and reduced risk of errors.

Conclusions:

• The proposed streamlined genotyping workflow offers significant advantages in speed, cost-effectiveness, and simplicity.
• Automated sequence analysis tools are effective for interpreting complex mutant data, suitable for bulk analysis.
• Academic and commercial laboratories are encouraged to adopt this improved genotyping method for increased efficiency.