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

Abstract

BackgroundWithin 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.ResultsWe 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.ConclusionWe 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.

Growing a circular economy with fungal biotechnology: a white paper

“…The molecular and analytical toolkit available for filamentous fungi has improved substantially during the 4 years since the first white paper was published in 2016 [4], demonstrating the high innovation potential of the filamentous fungal research community. Genome editing via CRISPR has become routine and is nowadays run in a multiplex manner for many reference strains [132,133]. Fourier-transformed infrared spectroscopy has become a next-generation phenotyping technology to identify fungal strains and their metabolic products [134], while the power of surface analysis tools, such as mass spectrometry-based imaging techniques, has been exploited to identify changes that are brought about by filamentous fungi and their enzymes when they attack and modify insoluble lignocellulose materials [135].…”

Upgrading of efficient and scalable CRISPR–Cas-mediated technology for genetic engineering in thermophilic fungus Myceliophthora thermophila

“…Although the CRISPR-Cas12a system has been intensively and successfully utilized in higher eukaryotes for genome editing [44][45][46][47][48][49], this system has been comparatively underexplored in microbes. Until now, Cas12a-based genomic editing tools have been demonstrated only in yeast, a few fungi species [50,51,57,58], and several bacteria [52][53][54][55], and have not yet been widely established in thermophilic filamentous fungi. While this manuscript was in revision, a functional CRISPR-Cpf1 system for gene editing was published in T. thermophilus (synonym of M. thermophila) [58] demonstrating that this system may work widely in filamentous fungi.…”

“…The crRNA used with Cas12a orthologs typically composed of a 23-25 nt guide sequence and a 19 nt direct repeat [43][44][45][46][47][48][49][50]. Thus far, the CRISPR-Cas12a system has been developed only in A. nidulans and A. niger [57], and T. thermophilus [58] as far as we know in filamentous fungi. To test whether the Cas12a effector can be used as an attractive alternative genome editing tool in thermophilic filamentous fungi, in this study, we firstly developed a new efficient CRISPR-Cas12a (AsCpf1) system in M. thermophila.…”

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine

“…One such approach involves CRISPR. Kwon and colleagues provided the first comprehensive analysis and evaluation of different CRISPR approaches for the modification of molds [62]. Guide RNAs were created and CRISPR nucleases were delivered to the filamentous fungus Thermothelomyces thermophilus on plasmids.…”