TKC-MC: An Effective Strategy for Generating Heritable Heterozygous Mutations in Essential Genes in Rice

Area of Science:

• Plant genetics
• Gene editing technologies
• Molecular biology

Background:

• CRISPR/Cas9 is vital for gene function studies and crop improvement.
• Essential genes require heterozygous mutations for plant survival and propagation.
• Standard CRISPR/Cas9 struggles to produce heterozygous mutants for essential genes in rice.

Purpose of the Study:

• To develop an efficient method for generating heritable heterozygous mutations in essential rice genes.
• To overcome limitations of traditional gene editing for essential gene research.

Main Methods:

• Designed Transgene-Killer CRISPR (TKC)-mediated mismatch-spacer targeting (TKC-M).
• Utilized a transgenic offspring self-elimination TKC platform with engineered gRNA-target mismatches.
• Tested single and dual mismatches at specific gRNA positions for varying target sensitivities.
• Employed a TKC-M Cocktail (TKC-MC) with multiple gRNA configurations.

Main Results:

• TKC-M successfully enriched heritable heterozygous and mosaic incomplete-edited T0 mutants.
• Single-base mismatches at positions 11 or 17 were effective for sensitive targets.
• Dual mismatches at positions 8 and 15 maximized heterozygotes for insensitive targets.
• TKC-MC significantly increased the incomplete-edited mutant ratio compared to standard TKC.

Conclusions:

• TKC-M provides an efficient strategy for generating heterozygous mutations in essential rice genes.
• This method facilitates research on essential genes and the creation of comprehensive mutant libraries.
• Establishes a technical foundation for plant genome-wide mutant library generation.