Figure 2.

Construction of SinLoG (Single Locus Glycolysis) strains IMX1770 and IMX1771 using the glycolysis swapping strategy.¹³ (A) A newly designed glycolysis is integrated in the CAN1 locus by simultaneous CRISPR/Cas9-aided editing of CAN1 and in vivo assembly of glycolytic expression cassettes and helper fragments (ARS418, ARS1211 and the selection marker HIS3). The > and < signs next to the gene names indicate the directionality of transcription and letters indicate the synthetic homologous recombination (SHR) sequence which was used for assembly. (B) Subsequently, the Single Locus Glycolysis present in the SGA1 locus was excised by double editing using CRISPR/Cas9 and replaced by the URA3 selection marker. The set of genes integrated in CAN1 is then the sole set of glycolytic genes present in the newly constructed strain and is essential for growth on glucose.