Practical guidance for the implementation of the CRISPR genome editing tool in filamentous fungi

Within the last years, numerous reports described successful application of the CRISPR nucleases Cas9 and Cpf1 for genome editing in filamentous fungi. However, still a lot of efforts are invested to develop and improve protocols for the fungus and genes of interest with respect to applicability, scalability and targeting efficiencies. These efforts are often hampered by the fact that—although many different protocols are available—none have systematically analysed and compared different CRISPR nucleases and different application procedures thereof for the efficiency of single- and multiplex-targeting approaches in the same fungus. We present here data for successful genome editing in the cell factory Thermothelomyces thermophilus, formerly known as Myceliophthora thermophila, using the three different nucleases SpCas9, FnCpf1, AsCpf1 guided to four different gene targets of our interest. These included a polyketide synthase (pks4.2), an alkaline protease (alp1), a SNARE protein (snc1) and a potential transcription factor (ptf1). For all four genes, guide RNAs were developed which enabled successful single-targeting and multiplex-targeting. CRISPR nucleases were either delivered to T. thermophilus on plasmids or preassembled with in vitro transcribed gRNA to form ribonucleoproteins (RNPs). We also evaluated the efficiency of single oligonucleotides for site-directed mutagenesis. Finally, we were able to scale down the transformation protocol to microtiter plate format which generated high numbers of positive transformants and will thus pave the way for future high-throughput investigations. We provide here the first comprehensive analysis and evaluation of different CRISPR approaches for a filamentous fungus. All approaches followed enabled successful genome editing in T. thermophilus; however, with different success rates. In addition, we show that the success rate depends on the respective nuclease and on the targeted gene locus. We finally present a practical guidance for experimental considerations aiming to guide the reader for successful implementation of CRISPR technology for other fungi.