Mix2seq on

All DNA fragments that constituted genetic elements used for plasmid or GTS assembly were amplified by polymerase chain reaction (PCR) using Phusion U Master Mix (ThermoFisher) according to the manufacturer’s recommendations. All PCR fragments used for cloning purposes were gel-purified using NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) by following the manufacturer’s protocol. All primers used in this study are denoted in Table_S1 and were purchased from Integrated DNA Technologies (IDT). GTSs with ∼400 bp upstream and ∼400 bp downstream targeting sequences for deletion of GOIs were constructed using the overlay PCR method ((26 (link)), see Supplementary Figure S1 for details). All plasmids were assembled by the uracil-specific excision reagent (USER™) method by cloning PCR fragments into AsiSI/Nb.bsmI cassette by following well-established protocols ((27 ), also see Supplementary Figures S2–S4 for details). Plasmid minipreps were prepared using GeneElute Plasmid MiniPrep Kit (Sigma-Aldrich) according to the manufacturer’s protocol. All newly constructed DNA constructs were validated by Sanger sequencing Mix2Seq ON (Eurofins). The full list of all plasmids used and constructed in this work is listed in Table_S2. All gene sequences were optimized using IDTs’ Codon Optimization Tool (https://eu.idtdna.com).

The obtained D. hansenii transformants were assessed by checking for expended phenotypes. In case of ade2, plates were visually checked by counting the number of red colonies with regard to the total number of colonies formed. Moreover, plates with sgRNA_ADE2-Cas9 transformants were also replica-plated using sterile velvet cloths on SC-ade medium to confirm adenine auxotrophy. The phenotypes of his4 or ura3 mutants were assessed by replica plating on SC-his and SC-ura media, respectively. Mutant strains showing the correct phenotype were tested by colony PCR to confirm introduction of the desired mutation(s). Routinely, 10 random colonies displaying the correct phenotype were selected for PCR analysis using primers designed to bind upstream and downstream of the genomic region containing the intended mutation(s) (see Supplementary Figure S5). All colony PCRs were done using Quick-Load® Taq 2X Master Mix according to the manufacturer’s recommendation. Eventually, PCR fragments of the expected size were column-purified using NucleoSpin Gel and PCR Clean-up Kit (MACHEREY-NAGEL) and sent for Sanger sequencing using Mix2Seq ON (Eurofins) service.