RNA-guided genome editing for target gene mutations in wheat

Area of Science:

• Plant biotechnology
• Molecular biology
• Genomics

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system is a powerful genome editing tool.
• Wheat (Triticum aestivum) possesses a large and complex genome, posing challenges for genetic modification.

Purpose of the Study:

• To apply CRISPR-Cas-mediated genome editing in wheat (Triticum aestivum).
• To investigate the efficacy of chimeric guide RNAs (cgRNA) for targeted mutagenesis in plant genes.

Main Methods:

• CRISPR-Cas9 system utilized with cgRNA targeting inositol oxygenase (inox) and phytoene desaturase (pds) genes.
• Experiments conducted in wheat cell suspension cultures and Nicotiana benthamiana leaves.
• Analysis of indel mutations, DNA fragment deletion, and target specificity.

Main Results:

• Indel mutations were successfully induced by cgRNA targeting single and multiple sites.
• Cas9 or sgRNA alone did not induce mutations.
• Duplex cgRNA targeting two sites in the same gene resulted in DNA fragment deletion.
• Multiplexing cgRNA allowed simultaneous targeting of two genes.
• Mismatches at the 3' end of the target site abolished cleavage, while 5' end mismatches reduced it.

Conclusions:

• CRISPR-Cas-mediated genome editing is effective for wheat improvement.
• cgRNA offers high target specificity, with potential to minimize off-target effects by selecting unique 3' end sequences.
• This technology provides a robust method for plant genome engineering.