Establishment of Efficient CRISPR-Cas9 PEG-Mediated DNA-Free Genome Editing Through Ribonucleoproteins Method in Hexaploid Sweetpotato (Ipomoea batatas L. (Lam)) Targeting the EIF-4E Genes

CRISPR-Cas9 technology has opened new perspectives in genome editing of clonally, asexually propagated and polyploid plants by enabling multiple allelic gene edits. Traditional <i>Agrobacterium</i>- and particle bombardment-mediated transformations, which rely on integration of gene-editing transgene cassettes, have been efficiently applied to several plants; however, concerns about the acceptability of resultant edited transgenic genotypes make these methods less attractive for vegetatively propagated crops. We leveraged and optimized the CRISPR-Cas9/sgRNA-RNPs system for delivery into protoplasts of the hexaploid sweetpotato cultivar PI-318846, targeting eukaryotic translation initiation factor isoform <i>4E</i> genes to enhance resistance to SPFMV potyviruses. To evaluate the efficiency of pre-assembled Cas9/sgRNA-RNP in sweetpotato transfection, single guide RNAs were designed to target putative host susceptibility genes: <i>IbeIF4E</i>, <i>IbeIF(iso)4E</i>, and <i>IbCBP</i>. Freshly isolated leaf protoplasts were subjected to CRISPR-CAS9-RNP PEG-mediated transfection under different parameters. Sweetpotato regenerants screened using PCR-RE-T7 assay, sequencing, and Inference CRISPR Edit analyses of target-site amplicons revealed the most efficient editing conditions utilizing 25% PEG with a 3:1 (15 µg:45 µg) ratio of Cas9/sgRNA-RNP for 25 min and 48 h incubation period. Different allelic InDels were obtained with editing efficiencies of 10-20% in regenerated plantlets, demonstrating that PEG-mediated CRISPR-RNP transfection system is key for advancing DNA-free editing tools in polyploid and vegetatively propagated crops.