Figure 3

Editing multiple genes in C. parapsilosis. (A) The URA3 gene in two strains of C. parapsilosis (CLIB214 and 90–137) was edited using pRIBO-URA3-385 and the relevant repair template (Supplementary Table S1). The figure shows a representative uracil auxotroph from each genetic background. Incorporation of the repair template was confirmed by sequencing. (B) The ADE2 gene was edited in one uracil auxotroph of C. parapsilosis CLIB214 (shown in panel A), using pRIBO-ADE2-D, generating strains that are pink on YPD, and fail to grow in the absence of uracil or adenine. Incorporation of the relevant repair template was confirmed by sequencing (Supplementary Fig. S3). (C) To edit CPAR2_101060, a repair template was designed including two stop codons, a unique 20 base pair tag, and a KpnI restriction site. The entire template is 108 base pairs. (D) Following transformation of two C. parapsilosis strains (CLIB214 and 90–137) colonies were screened by PCR using one common primer (CP101060_WT-R) and one primer specific for either the wild type (CP101060_WT_F, indicated here as WT-FWD) or the mutant (CP101060_MUT_F, indicated here as MUT-FWD) sequence, shown in brown and red in (C). A 458 bp PCR product was amplified from the mutant colonies only when the MUT_FWD primer was used, and from the wildtype (WT) only when the WT_FWD was used. (E) An 898 bp fragment was amplified using primers CP101060-F and CP101060_WT_R from two putative CPAR2_101060 disruptants from each C. parapsilosis background, and from wildtype C. parapsilosis CLIB214. Only strains in which CPAR2_101060 was edited were digested with KpnI. (F) To completely delete ADE2, a double stranded break was introduced using either sgADE2-B or sgADE2-D and a repair template including 40 bp from the 5′ and 3′ flanking regions and a unique 20 bp tag. Deletion mutants were verified by colony PCR using primers (DEL_FWD + REV) flanking the deleted region and this was confirmed by sequencing (Fig. S3).