In-locus gene silencing in plants using genome editing

Area of Science:

• Plant molecular biology
• Gene regulation
• Crop science

Background:

• Gene silencing is vital for developing desired crop traits.
• Current RNA interference (RNAi) methods face limitations like transgene instability.
• Upstream start codons (uATGs) in 5'UTRs can inhibit protein translation.

Purpose of the Study:

• To develop an efficient and robust gene silencing method in plants.
• To investigate the efficacy of introducing upstream start codon-containing elements (ATGEs) into plant genes.
• To enable tunable gene knockdown for crop improvement and biological studies.

Main Methods:

• Designed and introduced tailor-made ATGEs into the 5'UTR of target genes in plants.
• Utilized base editing to introduce uATGs, comparing its efficacy with ATGE insertion.
• Optimized ATGE design, including a bidirectional multifunctional ATGE4, for tunable gene knockdown.
• Applied the ATGE strategy to the Waxy gene involved in starch synthesis.

Main Results:

• Direct ATGE insertion adjacent to the primary start codon (pATG) effectively downregulated target protein expression.
• Base editing attempts to introduce uATGs showed limited success in silencing target genes.
• The ATGE strategy achieved consistent and substantial target protein downregulation.
• Tunable gene knockdown ranging from 19% to 89% was achieved using ATGE4.
• Introduction of ATGE into the Waxy gene resulted in reduced amylose content in grains.

Conclusions:

• Developed a programmable and robust method for gene knockdown in plants using ATGEs.
• The ATGE strategy overcomes limitations of traditional RNAi, offering greater control and stability.
• This method holds significant potential for exploring gene functions and enhancing crop traits.