Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• The CRISPR/Cas9 system offers efficient genome editing across diverse organisms.
• Previous studies on CRISPR/Cas9 in plants show varied success due to differences in target genes, expression systems, and tissues used.
• Standardized comparison of CRISPR/Cas9 components for plant genome editing is lacking.

Purpose of the Study:

• To evaluate and compare the mutation frequencies in rice using different Cas9 and guide-RNA (gRNA) expression cassettes under standardized conditions.
• To identify optimal Cas9/gRNA expression strategies for efficient targeted mutagenesis in rice.
• To develop an improved all-in-one CRISPR/Cas9 vector for rice genome editing.

Main Methods:

• Rice calli were transformed with various Cas9 and gRNA expression cassettes, introduced separately or sequentially.
• Mutation frequencies were assessed at specific endogenous target sequences.
• Different Cas9 expression cassettes and promoters (OsU6 vs. OsU3) for gRNA expression were evaluated.
• An all-in-one vector combining the most effective Cas9 and gRNA cassettes was constructed and tested.

Main Results:

• Mutation frequencies varied significantly based on the Cas9 expression cassette employed.
• A gRNA driven by the OsU6 promoter demonstrated superior performance compared to the OsU3 promoter.
• The optimized all-in-one expression vector significantly enhanced targeted mutagenesis frequency in rice calli.
• Bi-allelic mutant rice plants were successfully generated in the T0 generation.

Conclusions:

• The choice of Cas9 and gRNA expression cassettes critically impacts genome editing efficiency in rice.
• The OsU6 promoter is more effective for driving gRNA expression in rice compared to OsU3.
• An optimized all-in-one vector provides a highly efficient method for generating targeted mutations in rice.
• This standardized approach can be adapted to optimize CRISPR/Cas9 genome editing in other plant species.