A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genomics

Background:

• Precise genome editing is crucial for advancing biological research and crop improvement.
• Developing efficient and versatile gene editing systems for diverse plant species remains a key challenge.

Purpose of the Study:

• To establish and validate protoplast systems for precise DNA sequence modifications using preassembled Cas9 ribonucleoprotein (RNP) complexes.
• To develop reporter systems for sensitive detection of gene editing outcomes, including nonhomologous end joining (NHEJ) and homology-directed repair (HDR).

Main Methods:

• Utilized preassembled Cas9 ribonucleoprotein (RNP) complexes with dual guide RNAs (gRNAs) for gene disruption in plant protoplasts.
• Developed GFP reporter genes to quantify NHEJ and HDR efficiencies.
• Employed single-stranded oligodeoxynucleotide (ssODN) donors for precise gene editing via HDR.
• Applied preassembled primer editor (PE) RNPs for precise mutagenesis.

Main Results:

• Achieved up to ~90% indel efficiency for gene disruption in Arabidopsis protoplasts using Cas9 RNP and dual gRNAs.
• Demonstrated editing efficiencies up to 85% for NHEJ and 50% for HDR using GFP reporter systems.
• Obtained 7% precise editing of the AtALS gene via HDR using RNPs and ssODN donors.
• Reported 50% GFP reporter gene recovery and up to 4.6% editing frequency for a specific AtPDS mutation using PE RNPs.

Conclusions:

• CRISPR RNP variants in protoplasts offer a rapid, versatile, and efficient platform for plant gene editing.
• This system is valuable for developing, evaluating, and optimizing new gene editing tools and strategies.
• The established protoplast systems are applicable to a wide range of plant species for genetic manipulation.