High-throughput genome editing in rice with a virus-based surrogate system

Area of Science:

• Plant biotechnology
• Molecular biology
• Genomics

Background:

• Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies are widely used in plants.
• Large-scale genome editing in plants requires efficient and high-throughput methods.

Purpose of the Study:

• To develop a geminivirus-mediated surrogate system, Wheat Dwarf Virus-Gate (WDV-Gate), for high-throughput plant genome editing.
• To evaluate the efficiency of WDV-Gate for gene knock-out and sequence knock-in in rice.

Main Methods:

• Developed WDV-Gate using a transgenic rice recipient callus expressing Cas9 and a mutated hygromycin-resistant gene (HygM).
• Utilized a WDV-based construct for transient expression of two single guide RNAs (sgRNAs) targeting HygM and a gene of interest.
• Evaluated WDV-Gate on six rice loci, producing 874 T0 plants, and tested sequence knock-in for protein tagging.

Main Results:

• WDV-Gate significantly increased editing frequency (90.1% vs. 66.8%) and homozygous-edited plants (70.7% vs. 36.3%) compared to conventional methods.
• Plantlet regeneration efficiency showed a 2.31-fold increase with the WDV-Gate system.
• High editing frequency (94.4%) was achieved for large-scale editing of the SLR1 gene, and protein tagging via sequence knock-in reached up to 13% efficiency.

Conclusions:

• The WDV-Gate system, combining transient sgRNA expression and surrogate selection, is effective for high-throughput gene knock-out in plants.
• WDV-Gate facilitates efficient sequence knock-in for applications like protein tagging.
• This system offers a valuable tool for accelerating plant genome editing research and applications.