Figure 1
From: Gene editing in clinical isolates of Candida parapsilosis using CRISPR/Cas9
Autonomously replicating plasmids in C. parapsilosis. (A) pSAT1 was constructed by cloning a codon-optimized version of CAS9 between the promoter and terminator sequences of TEF1 from C. parapsilosis in a pUC57-based plasmid. SAT1 (nourseothricin resistance) expressed from the C. albicans ACT1 promoter was isolated from pSFS2A58, and ARS7, an autonomously replicating sequence from C. parapsilosis 43, was isolated from pGIZI. (B) CAS9 is expressed in C. parapsilosis cells transformed with pSAT1. RNA was isolated from four transformants and from one untransformed culture (UT). Expression of CAS9 and ACT1 was measured by RT-PCR. (C) pSAT1 is easily lost. Transformed cells were patched to YPD plates without nourseothricin (NTC) for 48 h, and then streaked on YPD and YPD + NTC. Colonies from YPD were repatched after 48 hr. All transformants lost nourseothricin resistance after just two passages.
