A CRISPR/Cas9-Based System with Controllable Auto-Excision Feature Serving Cisgenic Plant Breeding and Beyond

Area of Science:

• Agricultural Biotechnology
• Plant Molecular Biology
• Genome Editing

Background:

• Transgenic crops face regulatory hurdles due to biosafety concerns.
• Cisgenic crops present a publicly accepted alternative with improved regulatory review.
• Efficient tools are needed for cisgenic plant breeding.

Purpose of the Study:

• To develop a CRISPR/Cas9-based vector system for cisgenic plant breeding.
• To enable the delivery of a gene of interest (GOI) while removing genetic traces.
• To create novel cisgenic plant germplasms with desired traits.

Main Methods:

• Development of a CRISPR/Cas9 vector with auto-excision capabilities.
• Utilizing embedded multi-clonal sequences and inducible promoters for Cas9 control.
• Proof-of-concept study in *Arabidopsis thaliana*.

Main Results:

• Successful incorporation of a GOI and removal of selection markers and CRISPR/Cas9 components.
• Development of cisgenic plant germplasms within one to two generations.
• Heat treatment at 37°C improved editing efficiency to 100% with no off-target mutations.

Conclusions:

• The novel vector system is the first CRISPR/Cas9 tool for cisgenic plant breeding.
• This system facilitates the development of cisgenic plants with desired traits efficiently.
• The technology holds promise for future precision molecular breeding applications.