Fig. 1

Plasmids and cloning strategy. The backbone 1 (BB1) modules from the GoldernPiCS kit were used. Additional modules can be inserted into empty BB1 plasmids using BsaI Golden Gate Assembly (GGA) and fusion sites Fs1 (GGAG), Fs2 (CATG), Fs3 (GCTT), Fs4 (CGCT). A The genetic elements on BB1 plasmids were combined into a single transcription unit by BpiI GGA into a recipient crBB3_14 donor helper plasmid, containing homology regions for 04576, PFK1, or ROX1 target sites (uHR and dHR). The resulting plasmid was linearized and co-transformed with the corresponding CRISPi plasmid harbouring Cas9 and the sgRNA targeting the appropriate locus into K. phaffii host cells. B Multigene constructs can be prepared by assembling single transcription units into empty BB2 level plasmids using BpiI GGA. Fusion sites FsA (GATC), FsB (CCGG), FsC (AATT), etc. are then used to insert the expression cassettes into BB3 plasmids (e.g., crBB3_AC) using BsaI GGA