A novel recombinant CRISPR/Cas9 vector system for genome editing in plants

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 technology is widely used for gene knockout in plants.
• Low genome editing efficiency in plants remains a significant challenge for trait improvement.

Purpose of the Study:

• To construct a novel CRISPR/Cas9 binary vector cassette (pCR) for efficient plant genome editing.
• To enhance Cas9 expression and sgRNA cloning efficiency in plants.

Main Methods:

• Codon optimization of Cas9 cDNA with N-terminal and C-terminal nuclear localization signals.
• Incorporation of potato rcbS 5' UTR and removal of internal restriction sites in Cas9 gene.
• Design of a modular sgRNA cassette compatible with single or multiplexed cloning.
• Validation of the pCR vector system in *Nicotiana tabacum* and *Solanum tuberosum* via *Agrobacterium*-mediated transformation.

Main Results:

• The pCR vector system demonstrated efficient genome editing by successfully knocking out the phytoene desaturase (PDS) gene.
• Codon optimization and UTR incorporation improved Cas9 expression in plant cells.
• The sgRNA cassette design facilitated easy and versatile cloning of single or multiple sgRNAs.

Conclusions:

• The pCR binary vector system offers a versatile and efficient tool for plant genome editing.
• This system can be instrumental in improving agriculturally important plant traits through targeted gene modification.