Precision genome editing of GBSS using CRISPR/Cas9 RNP for starch improvement in Indian potato

Title : Precision genome editing of GBSS using CRISPR/Cas9 RNP for starch improvement in Indian potato

Abstract:

The potato (Solanum tuberosum L.) is the leading tuber crop and the third major food crop globally, after rice and wheat. India ranks as the second greatest producer of potatoes, farming over 50 types. Potato tubers are abundant in dietary carbohydrates, primarily as starch, which has extensive industrial applications. Starch comprises two primary polysaccharides, amylose and amylopectin, and their respective ratios dictate the functional and processing characteristics of starch. The Granule Bound Starch Synthase (GBSS) enzyme is crucial for amylose biosynthesis; thus, precise alteration of the GBSS gene can enhance starch quality for food processing industries, especially chips and French fries. In this study, we initially employed a CRISPR/Cas9 plasmid-based editing approach targeting the GBSS gene in Indian potato cultivars including Kufri Chipsona-1 (KC1), Kufri Fryo-M (KFM), and Kufri Frysona (KFS). Eight guide RNAs (gRNAs) targeting exon regions of the GBSS gene were designed and cloned into CRISPR/Cas9 expression vectors and introduced through Agrobacterium tumefaciens–mediated transformation. Multiple independent transgenic lines were generated and screened through genotyping PCR. Mutation analysis using Sanger sequencing and NGS revealed a relatively low editing efficiency of ~0.7% INDEL frequency, highlighting the challenges associated with plasmid-based editing in polyploid potato genomes. To address these constraints and generate transgene-free edited plants, we implemented a CRISPR/Cas9 ribonucleoprotein (RNP)-mediated genome editing approach, facilitating marker-free alteration of the GBSS gene. Delivery of preassembled Cas9-gRNA RNP complexes into potato protoplasts offers a viable strategy for effective editing and concurrent targeting of multiple alleles in tetraploid potato. Overall, this study develops marker-free low-amylose potato lines using CRISPR/Cas9-based and RNP-mediated genome editing. These methods will improve starch quality in Indian potato cultivars and contribute to producing high-value varieties for potato processing and molecular breeding.

Biography:

Payal Mavadiya is a plant molecular biologist with nearly ten years of research experience in gene editing, Agrobacterium-mediated transformation, and molecular cloning. During her doctoral research, she worked on the functional characterization of ACL5 homologs in plants, which involved gene cloning, generation of transgenic lines, detailed phenotypic characterization, and qRT-PCR–based transcript profiling. Currently, she is working as a Research Associate at the Gujarat Biotechnology Research Centre (GBRC) under the project titled “Genome Editing in Potato.” Her work focuses on CRISPR/Cas9-based genome editing for the development of marker-free edited lines, followed by molecular validation using Sanger sequencing and next-generation sequencing (NGS). Her strong interest in genome editing is driven by a desire to better understand plant physiology and the underlying cellular and molecular mechanisms that regulate plant growth and development.