Efficient genome editing in filamentous fungi via an improved CRISPR‐Cas9 ribonucleoprotein method facilitated by chemical reagents

Abstract

DNA double-strand break (DSB) repair induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9-CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X-100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP-based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi-target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on in vivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome-edited fungi for the food and medical industries, and has good prospects for commercialization.

Improving Hypoxia Adaption Causes Distinct Effects on Growth and Bioactive Compounds Synthesis in an Entomopathogenic Fungus Cordyceps militaris

“…Oligonucleotide primers were synthesized and sequenced by BioSune (Shanghai, China). All molecular cloning procedures, including genomic DNA extraction, DNA fragment acquisition, restriction-ligase reaction, transformation, colony verification, plasmid propagation, and sequencing, were operated according to the previous report ( Wang et al, 2020 ; Zou et al, 2020 ). The Pgpd promoter, Ptef promoter, and Sre1N encoding gene were amplified from the genomic DNA of CM01.…”

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

“…Transformed plasmids could also be degraded by endogenous nucleases into small DNA fragments, which may increase unwanted on-and off-target insertions in host cells [29]. In contrast, RNP-based genome editing is not limited by the efficiency of Cas9 and gRNA expression in vivo and it may protect gRNA from degradation [30]. As the RNPs are rapidly degraded after the transient exposure of the cells to Cas9, the chance of further rearrangements or off-target events that lead to unintended and nonspecific mutations is also lower [15].…”

“…Additionally, sequencing unraveled that the ura3 MUT_A had a 61 bp deletion near CRISPR site #119 (Figure S2A), suggesting that only one RNP was delivered to the nucleus during transformation. Efficiency of RNP delivery into fungal protoplasts with subsequent translocation to the nucleus is challenging and it has previously been reported to result in low genome editing efficiency in the ascomycete species Penicillium chrysogenum [15] and Trichoderma reesei [30]. The addition of surfactants is one option to improve cell membrane permeability during transformation, and Triton X-100 was shown to significantly increase the efficiency of RNP delivery in PEG-mediated transformation in T. reesei [30].…”

“…Efficiency of RNP delivery into fungal protoplasts with subsequent translocation to the nucleus is challenging and it has previously been reported to result in low genome editing efficiency in the ascomycete species Penicillium chrysogenum [15] and Trichoderma reesei [30]. The addition of surfactants is one option to improve cell membrane permeability during transformation, and Triton X-100 was shown to significantly increase the efficiency of RNP delivery in PEG-mediated transformation in T. reesei [30]. Furthermore, NHEJ has the disadvantage of introducing random insertions and translocations that affect neighboring genes [12].…”

Fast and efficient CRISPR-mediated genome editing in Aureobasidium pullulans using Cas9 ribonucleoproteins

“…RNPs enable immediate transient gene editing with reduced off-target effects (Kim et al , 2014; Yip, 2020). Cas9-sgRNA RNPs have been shown to efficiently edit the genomes of human and animal cells (Cho et al , 2013; Kim et al , 2014; Chaverra-Rodriguez et al , 2018; Chen et al , 2019), plant cells (Park et al , 2019; Lee et al , 2020) and various fungi (Foster et al , 2018; Zou et al , 2020). In A. pullulans , CRISPR mediated genome editing was previously performed using plasmids (Zhang et al , 2019) but RNPS have not yet been used.…”