CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.

VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.