Fig. 3
From: CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus
Integration of Spcas9 D147Y P411T at the SPR3 locus in S. pastorianus CBS1483. a Schematic representation of the integration of Spcas9 D147Y, P411T [36] and the nat selection marker. The integration is directed by homology regions of 480-bp (HRL) and 506-bp (HRM) to complete the double cross over integration. b Verification of the construction of strain IMX1187. Presence of the integration fragment carrying Spcas9 and nat genes and of the SPR3 open reading frame and was checked in four transformants with primers 3750 and 9394 (lanes 1–4) and with primers 3750 and 10435 (lanes 5–8) respectively. The strain host strain CBS1483 was used as reference. The transformant in lanes 1 and 5 was renamed IMX1187. The lane labelled with L designated the position of the DNA ladder [Gene ruler DNA ladder Mix (ThermoFischer Scientific #SM0332)]. c 1−Mapping of the 150 bp Illumina sequencing reads of IMX1187 onto the reference genome of CBS1483 [4] at the SPR3 locus reveals about 120-fold coverage of the homology regions HRL and HRM while the average coverage is about 60-fold. 2—Mapping of the IMX1187 150 bp Illumina sequencing reads onto the integrated fragment containing the Spcas9 and nat genes reveals about 60-fold coverage of the casette. 3—Assembly graph of IMX1187 mapping on SPR3, Spcas9 and their paired reads using SPAdes [46]