CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato

Area of Science:

• Plant Biotechnology
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 is a leading genome editing tool for plants.
• Potatoes are a crucial industrial crop requiring advanced genetic modification techniques.

Purpose of the Study:

• To develop tri- or tetra-allelic mutant potato lines using CRISPR/Cas9.
• To achieve efficient knockout of the granule-bound starch synthase (gbss) gene in potatoes.

Main Methods:

• Designed and constructed CRISPR/Cas9 expression vectors targeting three regions in the gbss exon I.
• Utilized Agrobacterium-mediated transformation for introducing the CRISPR/Cas9 system into potato.
• Analyzed mutations using CAPS, Sanger sequencing, and iodine staining.

Main Results:

• Generated potato lines with multiple nucleotide substitutions and indels in the gbss gene.
• Achieved frameshift mutations leading to premature stop codons and gbss gene knockout.
• Confirmed successful gene knockout, resulting in an amylose-free phenotype.

Conclusions:

• CRISPR/Cas9-mediated multiple guide RNA mutagenesis is highly effective for potato genome editing.
• This method enables efficient generation of gbss-knockout potato plants.
• The resulting amylose-free potatoes have significant potential for industrial applications.