Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes

Area of Science:

• Plant genetics
• Molecular biology
• Agricultural science

Background:

• The Auxin/Indole-3-Acetic Acid (Aux/IAA) gene family is crucial for auxin signaling in plants.
• The rice gene OsIAA23 influences root and shoot development, but previous mutants were difficult to study.
• CRISPR/Cas9 technology enables precise gene editing for functional analysis.

Purpose of the Study:

• To generate and characterize new OsIAA23 mutants in rice using CRISPR/Cas9.
• To investigate the impact of different genetic backgrounds (japonica and indica) on OsIAA23 function.
• To understand the molecular mechanisms underlying phenotypic variation in OsIAA23 mutants.

Main Methods:

• CRISPR/Cas9 gene editing in japonica (Wuyunjing24) and indica (Kasalath) rice.
• Extensive genome re-sequencing to detect off-target mutations.
• Phenotypic analysis of root and shoot development, and fertility.
• RNA-sequencing (RNA-seq) to analyze gene expression and transcript variants.

Main Results:

• Osiaa23 mutants in Kasalath with a 13-amino acid deletion exhibited severe dwarfing, root defects, and reduced fertility.
• Osiaa23 mutants in Wuyunjing24 with a frameshift insertion showed minimal phenotypic changes and normal fertility.
• RNA-seq revealed de novo mosaic transcripts in Wuyunjing24 mutants, bypassing premature termination and preserving wild-type function.

Conclusions:

• Different genetic backgrounds and mutation types significantly influence gene function and phenotypic outcomes.
• A 13-amino acid deletion in Kasalath OsIAA23 is critical for normal plant development and fertility.
• Alternative splicing can mask the effects of CRISPR/Cas9-induced mutations, leading to unexpected preservation of wild-type phenotypes in rice.