ArticleMicrobial biotechnology2021
Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents.
Article in Microbial biotechnology, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 41 papers.
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Who cites it
41 citing papers in PubMed, 90 citations in OpenAlex.
- Optimization of CRISPR/Cas9-based multi-gene editing system in Aspergillus oryzae and its application in α-amyrin biosynthesis.World journal of microbiology & biotechnology · 2026Article
- Ethyl methanesulfonate mutagenesis in fungi: genetic mechanisms, applications, and implications for agricultural biotechnology.Archives of microbiology · 2026Review
- Unlocking the Potential for Genetic Engineering of the Straw-Degrading MushroomJournal of fungi (Basel, Switzerland) · 2026Article
- A High-Efficiency CRISPR-Cas9 Ribonucleoprotein Genome Editing System inJournal of fungi (Basel, Switzerland) · 2026Article
- CRISPR/Cas systems in fungal biotechnology: advancing high-value metabolite synthesis for industrial and food security applications.Archives of microbiology · 2026Review
- Recent developments in CRISPR/Cas9 genome editing research for edible fungiculture.Functional & integrative genomics · 2026Review
- Accumulation of the spontaneous and random mutations is causative of fungal culture degeneration.Fundamental research · 2026Article
- Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.Mycopathologia · 2025Article
- Kingdom-wide CRISPR guide design with ALLEGRO.Nucleic acids research · 2025Article
- Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii.Journal of microbiology (Seoul, Korea) · 2025Article
- Unravelling fungal genome editing revolution: pathological and biotechnological application aspects.Archives of microbiology · 2025Review
- Gene Editing inJournal of fungi (Basel, Switzerland) · 2025Review
- CRISPR/Cas9-assisted gene editing reveals that EgPKS, a polyketide synthase, is required for the biosynthesis of preussomerins in Edenia gomezpompae SV2.World journal of microbiology & biotechnology · 2025Article
- Simply cut out - Combining CRISPR/Cas9 RNPs and transiently selected telomere vectors for marker free-gene deletion inFrontiers in genome editing · 2025Article
- Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.Journal of fungi (Basel, Switzerland) · 2024Review
- AdvancingBioengineering (Basel, Switzerland) · 2024Review
- Current Advances in the Functional Genes of Edible and Medicinal Fungi: Research Techniques, Functional Analysis, and Prospects.Journal of fungi (Basel, Switzerland) · 2024Review
- The protein methyltransferase TrSAM inhibits cellulase gene expression by interacting with the negative regulator ACE1 in Trichoderma reesei.Communications biology · 2024Article
- Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes).mSystems · 2024Article
- An efficient CRISPR/Cas9 genome editing system based on a multiple sgRNA processing platform in Trichoderma reesei for strain improvement and enzyme production.Biotechnology for biofuels and bioproducts · 2024Article
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
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.
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