Heritable gene editing using FT mobile guide RNAs and DNA viruses.

Area of Science:

• Plant molecular biology
• Gene editing technologies
• Agricultural biotechnology

Background:

• Virus-induced gene editing (VIGE) offers rapid gene function identification but limitations exist.
• Current VIGE systems struggle with heritability due to viruses not reaching the shoot apical meristem (SAM).
• Stable inheritance of gene edits is crucial for crop improvement and genetic studies.

Purpose of the Study:

• To develop a heritable VIGE system in Arabidopsis thaliana.
• To overcome the limitation of non-heritable gene edits in VIGE systems.
• To investigate the potential of the Flowering Locus T (FT) gene in delivering sgRNAs to the SAM for stable transformation.

Main Methods:

• Developed a cotton leaf crumple virus (CLCrV)-mediated VIGE system targeting BRI1, GL2, PDS genes, and GUS transgene.
• Transformed Arabidopsis thaliana with Cas9 overexpression constructs.
• Fused FT mRNA with sgRNAs (FT strategy) to facilitate sgRNA delivery into the SAM.

Main Results:

• The FT strategy enabled effective gene editing in infected plants.
• Achieved heritable mutations in the next generation with an efficiency of 4.35-8.79%.
• Gene-edited offspring were free of CLCrV genome components.

Conclusions:

• The FT strategy successfully established a heritable VIGE system in Arabidopsis thaliana.
• This method bypasses the need for tissue culture and stable transformation.
• The CLCrV-mediated VIGE system combined with the FT strategy provides a robust tool for generating heritable mutants.