Development of a CRISPR/Cas9 RNPmediated genetic engineering system in Paecilomyces variotii

Abstract

A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperaturesabove 40°C, was isolated from a paper mill and investigated as a host for heterologous proteinproduction. To prevent environmental dissemination of spores, UV mutagenesis wasemployed to create a conidia-deficient strain, UM7. This strain underwent gene editing usingCas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promotersequences amplified from the genomic DNA of P. variotii (PH4, PP2, PS8, Ptub, Ptef1, and PgpdA),along with a signal sequence-tagged eGFP, flanked by 5’-upstream (336 bp) and3’-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP intoprotoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutantscarrying complete HR donor DNAs with the six different promoter sequences, indicatingsuccessful homology-directed repair (HDR). Evaluation of promoter strength revealedthat PgpdA was the most effective for intracellular GFP production; however, it resulted in negligibleextracellular GFP signal under all promoter conditions. A newly edited strain with anHDR integration module connecting PgpdA directly to eGFP, without the signal sequence, exhibitedenhanced GFP expression in both mycelial cells and culture broth, suggesting thatthe signal peptide negatively affect protein expression and secretion. This work representsthe first successful RNP-mediated gene editing in P. variotii, contributing to the applicationof this thermophilic fungus in protein production.