TABLE 1.
Applications of CRISPR-Cas9 genome engineering tool in filamentous fungi.
| Species | Cas9 expression (selection marker, promoter) | Transformation method | Editing method | Application | Reference |
| Phytophthora sojae | Human-optimized codons Cas9, G418, Ham34 | PMT1 | NHEJ/HDR | Single-gene disruption | Fang and Tyler, 2016 |
| Ustilago maydis | Codon-optimized Cas9, ip, Otef (modified tef1)/hsp70 | PMT | NHEJ | Single/double-gene disruption | Schuster et al., 2016 |
| A. fumigatus | Human codon-optimized Cas9, pyr4/hph, niiA/gpdA | PMT | HDR | Single/double-gene disruption | Zhang et al., 2016 |
| A. oryzae | Codon-optimized Cas9, pyrG, amyB | PMT | NHEJ | Single-gene disruption | Katayama et al., 2016 |
| Penicillium chrysogenum | Human codon-optimized Cas9, amdS, xlnA | PMT | NHEJ/HDR | Single-gene disruption | Pohl et al., 2016 |
| Candida albicans | Codon-optimized Cas9, Nat, ENO1 | Lithium acetate | NHEJ/HDR | Single-gene disruption | Min et al., 2016 |
| A. fumigatus | pFC332, pyrG, TetON | PMT | NHEJ/HDR | Single-gene disruption | Weber et al., 2017 |
| A. niger | pFC332, pyrG/hph, tef1 | PMT | NHEJ/HDR | Single-gene disruption | Kuivanen et al., 2016 |
| Ustilago maydis | B913, ip, Otef (modified tefl)/ hsp70 | PMT | NHEJ | Multiple-gene disruption | Schuster et al., 2018 |
| A. oryzae | Codon-optimized Cas9, niaD/pyrG | PMT | NHEJ | Double-gene disruption | Nakamura et al., 2017 |
| Myceliophthora thermophila | Codon-optimized Cas9, Bar, tef1 | PMT/AMT2 | NHEJ/HDR | Single/multiple-gene disruption | Liu et al., 2017 |
| Talaromyces atroroseus | pFC332, hph, tef1 | PMT | NHEJ | Single-gene disruption | Nielsen et al., 2017 |
| Aspergillus carbonarius | pFC332, hph, tef1 | AMT | NHEJ/HDR | Single-gene disruption | Weyda et al., 2017 |
| A. niger | pFC332, hph, tef1 | PMT | NHEJ | Single-gene disruption | Kuivanen et al., 2017 |
| Ganoderma lucidum | Codon-optimized Cas9, ura3, gpdA | PMT | NHEJ | Single-gene disruption | Qin et al., 2017 |
| Beauveria bassiana | Codon-optimized Cas9, gfp/ura5/bar, gpdA | PMT | NHEJ/HDR | Single/multiple-gene disruption | Chen et al., 2017 |
| Alternaria alternata | pFC332, pyr4/hph, gpdA | PMT | NHEJ | Single-gene disruption | Wenderoth et al., 2017 |
| Shiraia bambusicola | Codon-optimized Cas9, hph, TrpC | PMT | NHEJ | Single-gene disruption | Deng et al., 2017a |
| Shiraia bambusicola | Codon-optimized Cas9, hph, TrpC | PMT | NHEJ/HDR | Single-gene disruption | Deng et al., 2017c |
| Nodulisporium sp. | Codon-optimized Cas9, Bar, TrpC | PMT | NHEJ/HDR | Single-gene disruption | Zheng et al., 2017 |
| Mucor circinelloides | SpCas9, pyr4 | PMT | NHEJ/HDR | Single/double-gene disruption | Nagy et al., 2017 |
| Leptosphaeria maculans | Human codon-optimized Cas9, Ip/G418/hph, act1 | AMT | NHEJ | Single-gene disruption | Idnurm et al., 2017 |
| Sporisorium scitamineum | HCas9, Nat/Hph, gapd | AMT | HDR | Single-gene disruption | Lu et al., 2017 |
| A. niger | pCas9, hyg/pyrG | PMT | NHEJ | Single-gene disruption | Sarkari et al., 2017 |
| A. fumigatus | In vitro-assembled Cas9 RNPs, hyg | PMT | HDR | Single-gene disruption | Al Abdallah et al., 2017 |
| Magnaporthe oryzae | Codon-optimized Cas9, hyg | PMT | NHEJ/HR | Single/double-gene disruption | Foster et al., 2018 |
| M. thermophila | Codon-optimized Cas9, neo, tef1 | PMT | NHEJ | Single-gene disruption | Xu et al., 2018 |
| Rhizopus delemar | Codon-optimized Cas9, pyrF | Electroporation | NHEJ | Single-gene disruption | Bruni et al., 2018 |
| M. thermophila | Codon-optimized Spmae, hyg/bar/neo, tef1/gpdA/TrpC | PMT | HDR | Single/multiple-gene disruption | Gu et al., 2018 |
| A. niger | Codon-optimized Cas9, pyrG/ble, tRNA | PMT | HDR | Single-gene disruption | Song et al., 2018 |
| Penicillium decumbens | Cas9 RNPs, gpdA, ergA | PMT | HDR | Single/multiple-gene disruption | Grijseels et al., 2018 |
| Fusarium graminearum | Codon-optimized Cas9, fludioxonil, gpdA | PMT | NHEJ/HDR | Single-gene disruption | Gardiner and Kazan, 2018 |
| A. niger | pFC332, hph, tef1 | PMT | NHEJ | Single-gene disruption | Kuivanen and Richard, 2018 |
| C. albicans | SpCas9, Nat/Phloxine B | Electroporation | NHEJ | Double-gene disruption | Shapiro et al., 2017 |
| Aspergilli | pFC332, argB/pyrG, tef1 | AMT | HDR | Multiple-gene disruption | Nodvig et al., 2018 |
| Blastomyces dermatitidis | pFC332, hph, tef1 | AMT | NHEJ | Single/double-gene disruption | Kujoth et al., 2018 |
| A. niger | Codon-optimized Cas9, hph/amdS, glaA | PMT | NHEJ/HDR | Single-gene disruption | Zheng et al., 2019 |
| C. albicans | Codon-optimized Cas9, Nat, ENO1 | Electroporation | HDR | Single/double-gene disruption | Vyas et al., 2018 |
| P. sojae | Human-optimized codons Cas9, G418, Ham34 | PMT | NHEJ | Double-gene disruption | Miao et al., 2018 |
| F. oxysporum | pFC332, hph | PMT | NHEJ/HDR | Single-gene disruption | Wang et al., 2018 |
| Ustilaginoidea virens | Codon-optimized Cas9, G418, pdc/cbh1 | AMT/PMT | NHEJ | Multiple-gene disruption | Liang et al., 2018 |
| Cordyceps militaris | Codon-optimized Cas9, 5-FOA/blpR, tef1 | AMT/PMT | NHEJ/HDR | Single-gene disruption | Chen et al., 2018 |
| Cryptococcus neoformans | Codon-optimized Cas9, Ntc, tef1 | Electroporation | HDR | Single-gene disruption | Wang, 2018 |
| Sclerotinia sclerotiorum | Codon-optimized Cas9, hph, tef1 | PMT | NHEJ/HDR | Single-gene disruption | Li et al., 2018 |
| Aspergillus fumigatus | pFC332, pyrG, tef1 | PMT | NHEJ | Single-gene disruption | Matsuda et al., 2018 |
| Ustilago trichophora | Human-optimized codons Cas9, marker-free, U6 | PMT | NHEJ | Single-gene disruption | Huck et al., 2019 |
| Penicillium subrubescens | Codon-optimized Cas9, hph, tef1 | PMT | NHEJ | Single-gene disruption | Salazar-Cerezo et al., 2020 |
| A. niger | pFC332, hph, tef1 | PMT | HDR | Multiple-gene disruption | van Leeuwe et al., 2019 |
| M. thermophila | Codon-optimized Cas12a, neo/bar, U6 | PMT | NHEJ/HDR | Multiple-gene disruption | Liu et al., 2019 |
| A. niger | pFC332, argB/pyrG, tef1 | PMT | HDR | Single-gene disruption | Leynaud-Kieffer et al., 2019 |
| A. oryzae | Codon-optimized Cas9, pyrG, amyB | PMT | HDR | Single/double-gene disruption | Katayama et al., 2019 |
| Alternaria alternata | pFC332, hph, tef1 | PMT | NHEJ | Single-gene disruption | Igbalajobi et al., 2019 |
| A. niger | Codon-optimized Cas9, pyrG | PMT | NHEJ/HDR | Single/multiple-gene disruption | Kuivanen et al., 2019 |
| Mucor circinelloides | SpCas9, pyr4 | PMT | HDR | Multiple-gene disruption | Nagy et al., 2019 |
| Leptosphaeria maculans | Human codon-optimized Cas9, hph, act1 | AMT | NHEJ | Double-gene disruption | Darma et al., 2019 |
| F. fujikuroi | Codon-optimized Cas9, hph, U6/5SrRNA | PMT | HDR | Multiple-gene disruption | Shi et al., 2019 |
| Ashbya gossypii | Human-optimized codons Cas9, G418, tef1 | Electroporation | NHEJ | Single-gene disruption | Jimenez et al., 2019 |
| Duddingtonia flagrans | pFC332, hph, tef1 | PMT | HDR | Single-gene disruption | Youssar et al., 2019 |
| A. oryzae | Aspergillus-optimized codons Cas9, pyrG, U6 | PMT | HDR | Single-gene disruption | Chutrakul et al., 2019 |
| T. reesei | Codon-optimized Cas9, pyr4 | PMT | HDR | Single-gene disruption | Hao and Su, 2019 |
| M. thermophila | Codon-optimized Cas9, bar/neo, tef1 | PMT | HDR | Double-gene disruption | Li et al., 2020a |
| Glarea lozoyensis | Codon-optimized Cas9, neo, 5S rRNA | AMT | NHEJ/HDR | Single/multiple-gene disruption | Wei et al., 2020 |
| Talaromyces pinophilus EMU | Codon-optimized Cas9, hph, gdpA | PMT | NHEJ | Single-gene disruption | Manglekar and Geng, 2020 |
| A. niger | pCsR1, hph, tef1 | PMT | NHEJ/HDR | Single/multiple-gene disruption | Rojas-Sánchez et al., 2020 |
| M. thermophila | Codon-optimized Cas9, neo, trpC | PMT/AMT | HDR | Single/multiple-gene disruption | Li et al., 2020b |
| A. niger | pFC332, pyrG/amdS, PnaII/TPI | PMT | HDR | Single-gene disruption | Dong et al., 2020 |
| T. reesei | Codon-optimized Cas9, ura5, U6 | AMT | NHEJ | Single-gene disruption | Wu et al., 2020 |
| A. niger | pCas9, pyrG/hph, 5S rRNA | PMT | HDR | Double-gene disruption | Zhang et al., 2020 |
1AMT, Agrobacterium tumefaciens-mediated transformation; 2PMT, Protoplast transformation.