An efficient mRNA delivery system for genome editing in plants

Area of Science:

• Plant biotechnology
• Genome editing
• Molecular biology

Background:

• Transgene-free genome editing is vital for crop improvement, minimizing unintended genomic alterations.
• Current mRNA delivery methods for plant genome editing are often inefficient and difficult to implement.
• Efficient delivery systems are needed to advance the application of mRNA-based genome editing in plants.

Purpose of the Study:

• To develop and optimize an efficient mRNA delivery system for transgene-free genome editing in plants.
• To enhance the efficiency of various genome editing techniques, including knock-out and base editing, using mRNA delivery.
• To demonstrate the system's effectiveness in key crop species like rice and wheat.

Main Methods:

• Optimization of 5' untranslated regions (5'UTRs) and poly(A) tails of in vitro-transcribed (IVT) mRNAs.
• Coating mRNA with protamine for enhanced delivery via particle bombardment.
• Development of the v2_TMV/DEN2 mRNA delivery system.

Main Results:

• The v2_TMV/DEN2 system significantly improved knock-out, A-to-G, and C-to-T base editing efficiencies (4.7-, 3.4-, and 2.5-fold increases, respectively) compared to plasmid delivery.
• Achieved high editing efficiencies in rice (5.0-180.8%) and wheat (26.1-26.2%) using the optimized mRNA system.
• Demonstrated substantially higher editing rates in rice suspension cells and wheat immature embryos 48 hours post-transformation.

Conclusions:

• The v2_TMV/DEN2 system offers a convenient and highly efficient method for transgene-free genome editing in plants.
• This optimized mRNA delivery approach overcomes previous limitations, paving the way for broader applications in crop improvement.
• The study provides a valuable tool for researchers seeking to advance plant genetic engineering through efficient and precise genome editing.