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GLABRA2-based selection efficiently enriches Cas9-generated nonchimeric mutants in the T1 generation
Xiangjiu Kong1, Wenbo Pan2, Nengxu Sun1
1The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China.
Plant Physiology
|October 5, 2021
Summary
A new GLABRA2 mutation-based visible selection (GBVS) system enhances CRISPR/Cas9 genome editing efficiency in Arabidopsis. This system significantly increases the identification of nonchimeric mutants, saving time and labor in plant research.
Area of Science:
- Plant molecular biology
- Genetics
- Biotechnology
Background:
- CRISPR/Cas9 is a key tool for plant genome editing.
- Egg cell-specific promoters in Arabidopsis reduce chimeras but yield low mutation rates.
- Efficient screening for nonchimeric mutants is crucial for genetic studies.
Purpose of the Study:
- To establish and evaluate a GLABRA2 mutation-based visible selection (GBVS) system for CRISPR/Cas9 in Arabidopsis.
- To improve the efficiency of identifying homozygous or biallelic mutants in the T1 generation.
- To reduce the time and labor required for obtaining specific mutant lines.
Main Methods:
- Development of a CRISPR/Cas9 system utilizing egg cell-specific promoters.
- Integration of the GLABRA2 gene for visible selection of nonchimeric mutants.
- Application of the GBVS system for screening T1 transformants in Arabidopsis thaliana.
Main Results:
- The GBVS system increased mutation screening frequency by 2.58- to 7.50-fold.
- 25%-48.15% of T1 plants selected via GBVS were homozygous or biallelic mutants.
- Nonchimeric triple mutants (pyr1/pyl1/pyl2) were obtained in the T1 generation at a 26.67% ratio using GBVS.
- Mutant phenotypes in T2 generation were not significantly affected by the glabrous background.
Conclusions:
- The GBVS system significantly enhances the efficiency of obtaining homozygous/biallelic and nonchimeric mutants in Arabidopsis.
- This method offers a time- and labor-saving alternative to existing CRISPR/Cas9 approaches.
- GBVS facilitates the rapid generation of complex polygenic mutants for research.
