Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9

Area of Science:

• Plant molecular biology
• Genome editing
• Biotechnology

Background:

• Targeted gene knockouts are established in plants using sequence-specific nucleases.
• Efficient gene fragment replacement and specific locus insertion in plant genomes remain challenging.
• The non-homologous end joining (NHEJ) pathway is a key mechanism in plant DNA repair.

Purpose of the Study:

• To develop efficient intron-mediated site-specific gene replacement and insertion methods in plants.
• To utilize the CRISPR-Cas9 system for precise genomic modifications.
• To assess the efficiency and heritability of generated mutations.

Main Methods:

• Employed a CRISPR-Cas9 system with single guide RNAs (sgRNAs) targeting adjacent introns.
• Utilized donor DNA templates containing sgRNA target sites for gene replacement and insertion.
• Applied these methods to the rice endogenous 5-enolpyruvylshikimate-3-phosphate synthase (OsEPSPS) gene.

Main Results:

• Achieved site-specific gene replacement in the OsEPSPS gene at a frequency of 2.0%.
• Obtained targeted gene insertions at a frequency of 2.2% using a single sgRNA targeting an intron.
• Generated glyphosate-resistant rice plants with intended OsEPSPS gene substitutions, which were heritable.

Conclusions:

• Developed efficient intron-mediated strategies for site-specific gene replacement and insertion in rice.
• Demonstrated the utility of these methods for creating herbicide-resistant crops.
• These approaches offer broad applicability for targeted genomic modifications in various plant species.