Single and multiple gene knockouts by CRISPR-Cas9 in maize

Area of Science:

• Plant Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 is a powerful genome editing tool widely used in various plant species.
• Targeted mutagenesis is crucial for understanding gene function and crop improvement.

Purpose of the Study:

• To demonstrate the efficiency of CRISPR-Cas9 for generating single and multiple gene knockouts in maize.
• To evaluate the utility of different CRISPR-Cas9 vector strategies for maize mutagenesis.

Main Methods:

• Generation of 12 plasmids with 28 single guide RNAs (sgRNAs) targeting 20 maize genes.
• Stable transformation of maize plants with CRISPR-Cas9 vectors.
• Analysis of mutant alleles using Sanger sequencing.

Main Results:

• Mutant alleles were obtained for 18 out of 20 targeted genes.
• 19% of mutant plants exhibited biallelic mutations; small insertions/deletions were common.
• Double and triple mutants were generated in a single step with minimal off-target effects.

Conclusions:

• CRISPR-Cas9 is a robust and efficient tool for targeted mutagenesis in maize.
• The technology facilitates the generation of complex mutants for functional genomics.
• High transmission rates of mutations to the next generation were observed.