# A Stable and Rapid Protoplast-Based CRISPR/Cas9-Ribonucleoprotein Complex Screening System for Octoploid Strawberry (Fragaria × ananassa)

## TL;DR

This paper introduces a new DNA-free CRISPR/Cas9 system for editing the complex genome of octoploid strawberries using protoplasts.

## Contribution

A DNA-free CRISPR/Cas9 protoplast system for efficient genome editing in octoploid strawberries is developed and optimized.

## Key findings

- Optimized protoplast isolation from fully developed leaves using specific enzyme combinations and culture conditions.
- Transient transfection efficiency reached up to 52.5% using 40% PEG 4000 and 20 min incubation.
- Successful DNA-free CRISPR/Cas9 editing of FaPDS and FaPG1 genes via protoplast-based RNP delivery.

## Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a simple and powerful tool, which enables gene knockout or insertion of new gene cassettes. This method has been applied to various plants and is used for crop improvement. Cultivated strawberry (Fragaria
× ananassa), a member of the Rosaceae family, is a high-value horticultural crop. However, its complex octoploid genome poses challenges for precise genome editing in polyploids. This study aimed to establish a protoplast-based, DNA-free genome-editing approach in the cultivated octoploid strawberry. We optimized the culture conditions and enzyme combinations to enable efficient protoplast isolation from the fully developed leaves. The highest protoplast yield was obtained with a Murashige and Skoog medium containing 1% sucrose and 2 mg/L 6-benzylaminopurine (BA), along with enzymatic digestion using 2% Viscozyme, 1% Celluclast, and 1% Pectinex. Transient transfection conditions were optimized using a green fluorescence protein (GFP) plasmid with the highest expression efficiency (up to 52.5%) observed using 40% PEG 4000 and 20 min incubation. Under these conditions, Cas9 ribonucleoproteins (RNPs) targeting the FaPDS and FaPG1 genes were introduced, and guide RNA (gRNA) screening was conducted by targeted deep sequencing. In conclusion, this study successfully demonstrated protoplast isolation and DNA-free CRISPR/Cas9 genome editing in cultivated strawberry. The optimized protoplast-based system provides a valuable platform for functional genomics and molecular breeding efforts in octoploid strawberries.