Transgene-Killer-CRISPR version 2 (TKC2) eliminates occasional transgene escape by coupling with a RUBY reporter

Area of Science:

• Plant biotechnology
• Gene editing technologies
• Molecular biology

Background:

• CRISPR-Cas9 technology enables precise gene editing in plants.
• Removing CRISPR transgenes from edited plants (T0) and progenies is crucial but challenging.
• Previous Transgene Killer CRISPR (TKC) methods showed incomplete transgene elimination in some T1 plants.

Purpose of the Study:

• To develop more efficient systems for generating transgene-free gene-edited plants.
• To improve upon existing Transgene Killer CRISPR (TKC) technology.
• To create a visually trackable system for transgene elimination.

Main Methods:

• Integrated Cas9, guide RNA (gRNA), and the RUBY reporter gene (RUBY-CRISPR) for monitoring.
• Developed four RUBY-TKC (TKC2) systems by combining RUBY-CRISPR with TKC variants.
• Tested TKC2 systems for efficiency in eliminating transgenes in T0 and subsequent generations.

Main Results:

• TKC2 systems demonstrated significantly higher efficiency in generating transgene-free edited progenies compared to non-TKC, TKC alone, and RUBY-CRISPR.
• Achieved up to 100% transgene-free edited progenies in the T0 generation.
• TKC2 systems allow for visualization of any escaped transgene-containing plants in later generations.

Conclusions:

• The developed TKC2 systems provide a highly effective and straightforward method for generating transgene-free gene-edited plants.
• These systems simplify rice functional genomics and genetic improvement by eliminating the need for extensive screening.
• TKC2 technology holds potential for broad application in gene editing across various crop species.