Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector.

Area of Science:

• Plant biotechnology
• Molecular biology
• Genetics

Background:

• CRISPR-Cas systems have transformed genome editing across organisms.
• Traditional plant CRISPR-Cas methods often require genetic transformation for reagent delivery.
• Optimizing synthetic guide RNAs (sgRNAs) for specific targets is crucial but time-consuming.

Purpose of the Study:

• To engineer a Potato virus X (PVX)-based vector for efficient delivery of multiple sgRNAs into adult solanaceous plants.
• To establish a virus-induced genome editing system for rapid multiplex gene editing.
• To assess the efficiency and heritability of genome editing using the PVX vector.

Main Methods:

• Engineered the Potato virus X (PVX) to express multiple synthetic guide RNAs (sgRNAs).
• Utilized the PVX vector for virus-induced genome editing in adult Nicotiana benthamiana plants expressing Streptococcus pyogenes Cas9.
• Analyzed gene editing efficiency by quantifying indels and assessing heritable mutations in progeny.

Main Results:

• The PVX vector enabled efficient expression of sgRNA arrays in adult solanaceous plants.
• Achieved nearly 80% indel formation in targeted Nicotiana benthamiana genes within days.
• Demonstrated highly efficient multiplex editing and obtained virus-free edited progeny with heritable biallelic mutations.

Conclusions:

• The developed PVX vector provides a fast, easy, and efficient method for multiplex CRISPR-Cas genome editing in adult plants.
• This tool is valuable for functional gene analysis and precision breeding, especially in Solanaceae crops.
• Virus-induced genome editing using PVX offers a promising alternative for accelerating crop improvement.