Etiolation promotes protoplast transfection and genome editing efficiency

Area of Science:

• Plant biotechnology
• Molecular biology
• Agricultural science

Background:

• DNA-free genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)-ribonucleoprotein (RNP) offers advantages like avoiding transgene integration and reducing off-target mutations.
• Optimizing protoplast transfection is crucial for maximizing the efficiency of CRISPR-RNP-mediated gene editing in plants.

Purpose of the Study:

• To investigate the impact of etiolation (darkness exposure) on the transfection efficiency of plant protoplasts.
• To determine if etiolation enhances DNA-free genome editing using CRISPR-RNP in lettuce and Chinese cabbage.

Main Methods:

• Plant seedlings (lettuce and Chinese cabbage) were cultivated under non-etiolated, etiolated, and de-etiolated conditions.
• Polyethylene glycol (PEG)-mediated transfection was used to assess green fluorescent protein (GFP) expression after etiolation.
• CRISPR-RNP delivery was performed on etiolated protoplasts for editing endogenous genes (LsPDS, LsFT), with editing efficiency quantified by targeted deep sequencing.

Main Results:

• Etiolated protoplasts exhibited significantly higher transfection efficiency, with 3.1-fold (lettuce) and 4.8-fold (Chinese cabbage) improvements in GFP expression compared to non-etiolated protoplasts.
• Gene editing efficiency using CRISPR-RNP was highest in etiolated protoplasts for both species.
• Editing efficiency for LsPDS and LsFT genes in lettuce showed an 8.7-fold and 4.4-fold improvement, respectively, in etiolated protoplasts.

Conclusions:

• Etiolation during seedling growth is an effective strategy to enhance protoplast transfection efficiency.
• Darkness exposure improves DNA-free genome editing outcomes in plant protoplasts, offering a promising method for crop improvement.