Fig. 5.

CRISPR-Cas9 mediated genome-editing of CAN1 gene in Y. lipolytica. (A) Design of single guide RNA (sgRNA) with type II promoter (TEF) driving the expression of CAN1 single guide RNA. CAN1: gRNA that target the arginine permease; HHR: hammer-head ribozyme; tracrRNA: trans-activating CRISPR RNA. (B) Secondary structure of the transcribed sgRNA flanked by upstream hammer-head ribozyme and downstream HDV ribozyme. Red arrow indicates the self-cleavage site. (C) PCR product fragments of the mutant CAN1 analyzed by gel electrophoresis. (D) CAN1 mutation efficiency in both transient sgRNA delivery and plasmid-based sgRNA delivery. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)