Optimization of CRISPR/Cas9-mediated CtPDS knockout in guar protoplasts

Area of Science:

• Plant Biotechnology
• Genomics
• Agricultural Science

Background:

• Guar (Cyamopsis tetragonoloba L. Taub.) is a climate-resilient legume with significant industrial and agricultural value.
• Gene editing is a crucial tool for crop improvement, yet its application in guar remained undocumented.
• There is a growing need for advanced genetic tools to enhance guar's valuable traits.

Purpose of the Study:

• To establish the first optimized protocol for CRISPR/Cas9 genome editing in guar using protoplasts.
• To demonstrate the efficiency and reliability of this gene editing system in guar.
• To lay the foundation for functional genomics and targeted trait improvement in guar.

Main Methods:

• Optimized isolation of intact and viable guar protoplasts from 6-day-old seedling cotyledons using the tape-sandwich method.
• Enhanced protoplast release and viability through enzymatic digestion (cellulase RS, pectinase) and vacuum infiltration.
• Developed polyethylene glycol (PEG)-mediated transformation protocol with high transient expression efficiency (∼57%) using a GFP plasmid and specific promoter.

Main Results:

• Achieved 100% editing efficiency in guar protoplasts targeting the Cyamopsis tetragonoloba phytoene desaturase (CtPDS) gene.
• Confirmed highly efficient editing via PCR amplification and Sanger sequencing, showing fragment deletions of 714–1061 bp in CtPDS.
• Demonstrated successful CRISPR/Cas9-induced mutations in all transformed samples.

Conclusions:

• Established a reliable, transient protoplast system for CRISPR/Cas9 genome editing in guar.
• This protocol enables functional genomics studies and targeted trait improvement in guar.
• Provides a critical foundation for future advancements in guar crop improvement through gene editing.