Chemicals, Reagents, Oligodeoxyribonucleotides, and Cloning

All medium components and chemicals were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands) or Merck (Darmstadt, Germany). Oligodeoxyribonucleotide primers were obtained from Merck. Enzymes were obtained from Thermo Fisher Scientific (Waltham, MA) unless otherwise stated. For the design of nucleic acid constructs, in silico restriction cloning, and inspection of Sanger sequencing results, SnapGene (GSL Biotech) was used. PCR amplifications were conducted using KAPA HiFi HotStart ReadyMix (Roche Diagnostics, Rotkreuz, Switzerland). Templates for PCR amplifications were acquired from various sources (Table 1) or ordered as synthetic DNA fragments from Thermo Fisher Scientific. All internal BpiI and BsaI cloning sites (and in some cases DraIII and Esp3I) were removed during cloning from the DNA fragments, and these sequences were manually curated for frequent codons in P. rubens. All of the vectors were constructed using the MoClo assembly system and protocol.¹⁹ The receiver backbones (established in the Modular Cloning assembly¹⁹) used for constructing the genetic parts containing entry vectors are highlighted in Figure ​Figure11b. As the linker sequences between the genetic parts in the transcription unit are based on the standard MoClo language (Figure ​Figure11a), the parts are compatible with modular systems that use this linker system.

Correctly assembled plasmids were identified with blue/white screening and confirmed by sequencing. The transcription unit expressing SpCas9–eGFP-NLS on a fungal shuttle vector (pLM-AMA002_P40s-dSpCas9-eGFP-NLS-Ttif35) was assembled using a mixture of 30 fmol of each entry vector (P40s An0465 (P1), dSpCas9(m2) (CDS2), eGFP-NLS (CDS5), and Ttif35 (T1)) and the backbone vector pLM-AMA002.

The 50% shorter AMA1 sequence²⁵ was created by PCR and integrated into a MoClo entry vector. The autonomously replicating shuttle vector carrying the AMA1 sequence was based on the pDSM-JAK-109 backbone where the pGpda-DsRed-SKL-TpenDE transcription unit was removed using the BspTI and NotI restriction enzymes. The linear vector was treated with the Klenow Fragment of DNA polymerase I and self-ligated into a circular vector using the T4 DNA ligase according to the instructions of the manufacturer, creating a new AMA1 vector without DsRed expression. This vector was cloned with a removable LacZ gene cloning site using BspTI, based on the “level 1” receiver backbones of the MoClo system, to create pLM-AMA002.