Developed a viral agent screening system to achieve efficient knockout and knock-in of rice genes

Recently, Lu Yuming's research group and Academician Zhu Health's team of Shanghai Jiao Tong University published a research paper entitled "High-throughput genome editing in rice with a virus-based surrogate system" in the Journal of Integrative Plant Biology (https?:/ /doi.
org/10.
1111/jipb.
1?3381）
。 In this study, a high-throughput gene knockout and knock-in system
for rice was established by efficiently delivering and expressing sgRNA by twin viruses, combined with rice receptor materials stably expressing Cas9 and hygromycin agent screening systems.

In recent years, with the widespread application of gene editing technology in plant breeding research, more and more research has focused on using gene editing technology to saturate mutations in the promoter or domain of the target gene to create genetic diversity
.
However, due to the huge workload of traditional T-DNA-based plant genetic transformation and low editing efficiency, large-scale saturation mutation has a high cost and is difficult to widely apply
.

In this study, two sgRNAs were efficiently delivered and expressed in a rice background material with high expression Cas9 and hygromycin agent screening system, one of which was an sgRNA-targeted hygromycin agent screening system to efficiently repair the inactivated hygromycin gene (HygM) for screening transient editing events.
Another sgRNA targets the gene of interest, enabling efficient editing, and the system is named WDV-Gate03
.
Compared with the traditional T-DNA system, the WDV-Gate03 system can achieve efficient tissue culture screening without T-DNA integration, which greatly improves the number of seedling regeneration.
At the same time, the efficiency of gene editing (66.
8% vs.
90.
1%) and homozygous editing (including biallelel mutations, 36.
3% vs.
70.
7%) has also been greatly improved, which shows the feasibility
of WDV-Gate system for high-throughput gene editing.

The team then designed 42 targets to target the DELLA domain of the SLR1 gene to saturate it
.
The identification results showed that the editing efficiency of SLR1 mutant library was as high as 94.
4%, and multiple allele mutation types
of different plant heights were generated.
Further analysis found that most of these regenerated plants (68.
9%) did not contain T-DNA, showing the unique advantages
of the system in high-throughput editing of rice.
Previously, the research team used chemically modified donor DNA to achieve efficient targeted knock-in and replacement
of segments of the rice genome.
In this study, the authors combined chemically modified donor DNA with the WDV-Gate03 system to precisely fuse 3xFlag tags at the N-terminus of AGO4, MDH2, and TOR genes, with tag in situ fusion efficiency of up to 13% and stable inheritance to offspring
.
This result further demonstrates the potential
of the system for fragment knock-in.

Professor Lu Yuming, College of Agriculture and Biology, Shanghai Jiao Tong University, and Academician Zhu Health of Southern University of Science and Technology are the co-corresponding authors of this paper, and Tian Yifu, Zhong Dating and Li Xinbo, Research Center for Plant Stress Biology, Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences, are co-first authors
.
This research was supported
by the National Natural Science Foundation of China and the National Key Research and Development Program.

Author:

College of Agriculture and Biology

Feeds:

College of Agriculture and Biology