Optimization of In Vitro CRISPR/Cas9 Test for Targeting Virescens Gene in Oil Palm

Abstract

The CRISPR/Cas9 has been used to target Virescens gene for fruit color development during the ripening process in oil palm. When ripped, Virescens fruits turn into bright red-orange from green. The development of Virescens fruits is important to help the harvesters determine fruit’s maturity and ripening. The Virescens gene has more than one sgRNA and each of them has different cleavage efficiency. The in vitro screening is widely used for study different sgRNA efficiency. However, the sgRNA elicits partial cleavage that could result from failure to form an intact secondary structure. Here we studied the modification of tracrRNA sequences and heat treatments that might contribute to an intact sgRNA structure. By adding 5 bp at the upper stem, the knockout efficiency is improved. In contrast, the combination of extended-upper stem and poly-U changes at the lower stem decreases the cleavage efficiency. In addition, the heat treatment causes misfolding of sgRNA, reducing the cleavage efficiency. The nucleotide compositions might cause the interaction within sgRNA, resulting in low cleavage efficiency. Further studies in the structural interaction of modified sgRNA should be conducted to emphasize this study.