Potato processing suffers from a high glycemic index due to amylopectin-rich starch and from undesirable color and acrylamide formation during frying, mainly caused by cold-induced sweetening (CIS). To address both issues simultaneously, we used CRISPR-Cas9 to knock out two key genes in diploid Solanum chacoense: ScSBE II (starch branching enzyme II), which controls amylopectin biosynthesis, and ScVInv (vacuolar invertase), a central regulator of CIS. Knockout of ScSBEⅡ increased tuber fresh weight-based absolute amylose content by ∼5-fold versus wild type. Amylose proportion in total starch elevated from 22% to 54%, while amylopectin abundance declined 1.5-fold, substantially optimizing the amylose/amylopectin mass ratio of tuber starch. These lines also showed a 4-fold reduction in rapidly digestible starch (RDS), 3.5‑fold and 1.2‑fold increases in slowly digestible starch (SDS) and resistant starch (RS), respectively, and markedly improved pasting properties. Enzyme assays confirmed a 2.5‑fold reduction in ScSBE II activity. In wild‑type (WT) tubers, cold storage (4 °C, 7 d) increased ScVInv activity ∼5‑fold (to 48 μg·min⁻¹·g⁻¹) and reducing sugars 6‑fold (from 11 to 68 mg·g⁻¹). Notably, ScVInv single‑knockout and ScSBE II/ScVInv double‑knockout lines produced chips with lighter color and much lower acrylamide than WT or ScSBE II single‑knockout lines. This dual‑gene editing strategy creates novel potato germplasm with enhanced resistant starch (health benefit) and superior processing quality (safer, visually appealing fried products).
