DNA-free CRISPR genome editing in raspberry (Rubus idaeus) protoplast through RNP-mediated transfection

Area of Science:

• Plant Biotechnology
• Genome Editing
• Soft Fruit Crop Improvement

Background:

• Protoplast systems facilitate genome editing without foreign DNA integration in plants.
• DNA-free editing preserves cultivar genetics and non-transgenic status.
• Raspberry advanced breeding is hindered by a lack of protoplast isolation and genome editing protocols.

Purpose of the Study:

• To establish a high-yielding protoplast isolation protocol for raspberry stem cultures.
• To implement DNA-free genome editing in raspberry protoplasts using CRISPR-Cas9 ribonucleoprotein complexes (RNPs).
• To demonstrate targeted mutagenesis in raspberry for precision breeding applications.

Main Methods:

• Developed a novel, high-yielding protoplast isolation method from raspberry stem cultures.
• Utilized pre-assembled CRISPR-Cas9 ribonucleoprotein complexes (RNPs) for efficient gene editing.
• Performed targeted mutagenesis on the phytoene desaturase gene within raspberry protoplasts.

Main Results:

• Achieved a 19% genome editing efficiency in raspberry protoplasts targeting the phytoene desaturase gene.
• Demonstrated that amplicon sequencing is the most sensitive method for detecting low-efficiency indels.
• Successfully established the first DNA-free genome editing protocol for raspberry protoplasts.

Conclusions:

• The developed protocol enables efficient DNA-free genome editing in raspberry protoplasts.
• This method is crucial for understanding gene function and advancing precision breeding in raspberries.
• The protocol offers a valuable platform for future genetic improvements in this soft fruit crop.