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High-Throughput CRISPR/Cas9 Mutagenesis Streamlines Trait Gene Identification in Maize.
Hai-Jun Liu1, Liumei Jian2, Jieting Xu1,3
1National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China.
The Plant Cell
|February 28, 2020
Summary
Researchers used CRISPR/Cas9 gene editing to rapidly identify important agronomic genes in maize (Zea mays). This high-throughput method successfully validated gene functions, accelerating crop improvement for complex plant genomes.
Area of Science:
- Plant genetics and genomics
- Agricultural biotechnology
- Gene editing technologies
Background:
- Maize (Zea mays) is a globally significant crop, yet its complex genome hinders the identification and utilization of genes for trait improvement.
- Cloning and functional validation of agronomically important genes in maize remain challenging due to genetic complexity.
Purpose of the Study:
- To develop and apply a high-throughput targeted mutagenesis system using CRISPR/Cas9 for efficient gene discovery in maize.
- To validate the function of candidate genes related to agronomic and nutritional traits in maize.
- To explore gene editing event profiles and repair mechanisms in a complex plant genome.
Main Methods:
- Integrated multiplexed CRISPR/Cas9 mutagenesis with genetic mapping and genomic analyses.
- Employed low-cost barcode-based deep sequencing for identifying gene editing events.
- Utilized an existing algorithm, originally developed for human cell lines, to predict gene editing profiles.
Main Results:
- Successfully targeted 743 candidate genes, with 412 edited sequences from 118 genes precisely identified in phenotypically altered individuals.
- Observed gene editing event profiles comparable to human cell lines, predictable by established algorithms.
- Documented frequent, unexpected homology-directed repair mediated by endogenous templates, potentially due to chromosomal proximity.
Conclusions:
- The integration of forward and reverse genetics through a targeted mutagenesis library enables rapid validation of essential agronomic genes in complex plant genomes like maize.
- This study provides a robust framework for high-throughput CRISPR experiments in plants, facilitating accelerated crop trait improvement.
- The findings offer insights into gene editing mechanisms and predictability in plants, paving the way for enhanced crop breeding strategies.
