Login | Register

Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger


Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger

Song, Letian, Ouedraogo, Jean-Paul, Kolbusz, Magdalena, Nguyen, Thi Truc Minh and Tsang, Adrian (2018) Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger. PLOS ONE, 13 (8). e0202868. ISSN 1932-6203

[thumbnail of Ouedraogo-PLOS-2018.pdf]
Text (application/pdf)
Ouedraogo-PLOS-2018.pdf - Published Version
Available under License Creative Commons Attribution.

Official URL: http://dx.doi.org/10.1371/journal.pone.0202868


As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in filamentous fungi to improve the efficiency of genome alteration. However, the method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Here we report a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. Co-transformation with DNA template for homologous recombination, the CRISPR/Cas9 system resulted ~42% efficiency of gene replacement in a strain with a functioning non-homologous end joining machinery (kusA+), and an efficiency of >90% gene replacement in a kusA- background. Our results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.

Divisions:Concordia University > Faculty of Arts and Science > Biology
Item Type:Article
Authors:Song, Letian and Ouedraogo, Jean-Paul and Kolbusz, Magdalena and Nguyen, Thi Truc Minh and Tsang, Adrian
Journal or Publication:PLOS ONE
  • Concordia Open Access Author Fund
  • NSERC Strategic Industrial Biocatalysis Network
Digital Object Identifier (DOI):10.1371/journal.pone.0202868
ID Code:984702
Deposited By: Krista Alexander
Deposited On:27 Nov 2018 19:51
Last Modified:27 Nov 2018 19:51


1. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, et al. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol. 2007;25: 221–231. pmid:17259976

2. Meyer V. Genetic engineering of filamentous fungi—Progress, obstacles and future trends. Biotechnol Adv. 2008;26: 177–185. pmid:18201856

3. Meyer V, Wanka F, van Gent J, Arentshorst M, van den Hondel CAMJJ, Ram AFJ. Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger. Appl Environ Microbiol. 2011;77: 2975–2983. pmid:21378046

4. Andersen MR. Elucidation of primary metabolic pathways in Aspergillus species: orphaned research in characterizing orphan genes. Brief Funct Genomics. 2014;13: 451–455. pmid:25114096

5. Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science. 2003;300: 764–764. pmid:12730594

6. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009;326: 1509–1512. pmid:19933107

7. Bellaiche Y, Mogila V, Perrimon N. I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. Genetics. 1999;152: 1037–1044. pmid:10388822

8. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci. 2012;109: E2579–E2586. pmid:22949671

9. Ouedraogo JP, Arentshorst M, Nikolaev I, Barends S, Ram AFJ. I-SceI enzyme mediated integration (SEMI) for fast and efficient gene targeting in Trichoderma reesei. J Biotechnol. 2016;222: 25–28. pmid:26860210

10. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186: 757–761. pmid:20660643

11. Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33: 543–548. pmid:25803306

12. Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol. 2015;33: 1162–1164. pmid:26479191

13. Port F, Chen H-M, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci. 2014;111: E2967–E2976. pmid:25002478

14. Nødvig CS, Nielsen JB, Kogle ME, Mortensen UH. A CRISPR-Cas9 system for genetic engineering of filamentous fungi. Yu J-H, editor. PLOS ONE. 2015;10: e0133085. pmid:26177455

15. Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015;81: 2506–2514. pmid:25636838

16. Sander JD, Joung JK. CRISPR-Cas systems for genome editing, regulation and targeting. Nat Biotechnol. 2014;32: 347–355. pmid:24584096

17. Katayama T, Tanaka Y, Okabe T, Nakamura H, Fujii W, Kitamoto K, et al. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol Lett. 2016;38: 637–642. pmid:26687199

18. Nødvig CS, Hoof JB, Kogle ME, Jarczynska ZD, Lehmbeck J, Klitgaard DK, et al. Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli. Fungal Genet Biol. 2018;

19. Zhang T, Gao Y, Wang R, Zhao Y. Production of guide RNAs in vitro and in vivo for CRISPR using ribozymes and RNA polymerase II promoters. BIO-Protoc. 2017;7.

20. White RJ. Transcription by RNA polymerase III: more complex than we thought. Nat Rev Genet. 2011;12: 459–463. pmid:21540878

21. Hopper AK, Pai DA, Engelke DR. Cellular dynamics of tRNAs and their genes. FEBS Lett. 2010;584: 310. pmid:19931532

22. Cieśla M, Towpik J, Graczyk D, Oficjalska-Pham D, Harismendy O, Suleau A, et al. Maf1 is involved in coupling carbon metabolism to RNA polymerase III transcription. Mol Cell Biol. 2007;27: 7693–7702. pmid:17785443

23. Male G, von Appen A, Glatt S, Taylor NMI, Cristovao M, Groetsch H, et al. Architecture of TFIIIC and its role in RNA polymerase III pre-initiation complex assembly. Nat Commun. 2015;6: 7387. pmid:26060179

24. Young LS, Rivier DH, Sprague KU. Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors. Mol Cell Biol. 1991;11: 1382–1392. pmid:1996100

25. Geiduschek EP, Tocchini-Valentini GP. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57: 873–914. pmid:3052292

26. Schiffer S, Rösch S, Marchfelder A. Assigning a function to a conserved group of proteins: the tRNA 3′‐processing enzymes. EMBO J. 2002;21: 2769–2777. pmid:12032089

27. Kruszka K, Barneche F, Guyot R, Ailhas J, Meneau I, Schiffer S, et al. Plant dicistronic tRNA–snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z. EMBO J. 2003;22: 621–632. pmid:12554662

28. Xie K, Minkenberg B, Yang Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci. 2015;112: 3570–3575. pmid:25733849

29. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013;41: 4336–4343. pmid:23460208

30. Jørgensen TR, Park J, Arentshorst M, van Welzen AM, Lamers G, vanKuyk PA, et al. The molecular and genetic basis of conidial pigmentation in Aspergillus niger. Fungal Genet Biol. 2011;48: 544–553. pmid:21277986

31. Storms R, Zheng Y, Li H, Sillaots S, Martinez-Perez A, Tsang A. Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger. Plasmid. 2005;53: 191–204. pmid:15848224

32. Aleksenko A, Clutterbuck AJ. The plasmid replicator AMA1 in Aspergillus nidulans is an inverted duplication of a low-copy-number dispersed genomic repeat. Mol Microbiol. 1996;19: 565–574. pmid:8830247

33. Ballance DJ, Turner G. Development of a high-frequency transforming vector for Aspergillus nidulans. Gene. 1985;36: 321–331. pmid:3000883

34. Goosen T, Bloemheuvel G, Gysler C, Bie DA de, Broek HWJ van den, Swart K. Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene. Curr Genet. 1987;11: 499–503. pmid:2836081

35. Hang YD, Woodams EE. Apple pomace: a potential substrate for citric acid production by Aspergillus niger. Biotechnol Lett. 1984;6: 763–764.

36. Li Y, Chooi Y-H, Sheng Y, Valentine JS, Tang Y. Comparative characterization of fungal anthracenone and naphthacenedione biosynthetic pathways reveals an α-hydroxylation-dependent claisen-like cyclization catalyzed by a dimanganese thioesterase. J Am Chem Soc. 2011;133: 15773–15785. pmid:21866960

37. Shrivastav M, De Haro LP, Nickoloff JA. Regulation of DNA double-strand break repair pathway choice. Cell Res. 2008;18: 134–147. pmid:18157161

38. Carvalho NDSP, Arentshorst M, Jin Kwon M, Meyer V, Ram AFJ. Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl Microbiol Biotechnol. 2010;87: 1463–1473. pmid:20422182

39. Meyer V, Arentshorst M, El-Ghezal A, Drews A-C, Kooistra R, van den Hondel CAMJJ, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol. 2007;128: 770–775. pmid:17275117

40. Zhang J, Mao Z, Xue W, Li Y, Tang G, Wang A, et al. Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger. Curr Microbiol. 2011;62: 1342–1346. pmid:21225265

41. Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M. Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun. 2014;5: ncomms6344.

42. Arazoe T, Miyoshi K, Yamato T, Ogawa T, Ohsato S, Arie T, et al. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotechnol Bioeng. 2015;112: 2543–2549. pmid:26039904

43. Zhang C, Meng X, Wei X, Lu L. Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genet Biol. 2016;86: 47–57. pmid:26701308

44. Schuster M, Schweizer G, Reissmann S, Kahmann R. Genome editing in Ustilago maydis using the CRISPR–Cas system. Fungal Genet Biol. 2016;89: 3–9. pmid:26365384

45. Pohl C, Kiel JAKW, Driessen AJM, Bovenberg RAL, Nygård Y. CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synth Biol. 2016;5: 754–764. pmid:27072635

46. Liu Q, Gao R, Li J, Lin L, Zhao J, Sun W, et al. Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering. Biotechnol Biofuels. 2017;10: 1. pmid:28053662

47. Sugano SS, Suzuki H, Shimokita E, Chiba H, Noji S, Osakabe Y, et al. Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system. Sci Rep. 2017;7: 1260. pmid:28455526

48. Matsu-ura T, Baek M, Kwon J, Hong C. Efficient gene editing in Neurospora crassa with CRISPR technology. Fungal Biol Biotechnol. 2015;2: 4. pmid:28955455

49. Fuller KK, Chen S, Loros JJ, Dunlap JC. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot Cell. 2015;14: 1073–1080. pmid:26318395

50. Brow DA, Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988;334: 213–218. pmid:3041282

51. Huang Y, Maraia RJ. Comparison of the RNA polymerase III transcription machinery in Schizosaccharomyces pombe, Saccharomyces cerevisiae and human. Nucleic Acids Res. 2001;29: 2675–2690. pmid:11433012

52. Schwartz CM, Hussain MS, Blenner M, Wheeldon I. Synthetic RNA polymerase III promoters facilitate high-efficiency CRISPR/Cas9-mediated genome editing in Yarrowia lipolytica. ACS Synth Biol. 2016;5: 356–359. pmid:26714206

53. Liu R, Chen L, Jiang Y, Zhou Z, Zou G. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov. 2015;1: celldisc20157.

54. Weyda I, Yang L, Vang J, Ahring BK, Lübeck M, Lübeck PS. A comparison of Agrobacterium-mediated transformation and protoplast-mediated transformation with CRISPR-Cas9 and bipartite gene targeting substrates, as effective gene targeting tools for Aspergillus carbonarius. J Microbiol Methods. 2017;135: 26–34. pmid:28159628

55. Huertas P. DNA resection in eukaryotes: deciding how to fix the break. Nat Struct Mol Biol. 2010;17: 11–16. pmid:20051983

56. Meyer V, Fiedler M, Nitsche B, King R. The Cell Factory Aspergillus Enters the Big Data Era: Opportunities and Challenges for Optimising Product Formation. In: Krull R, Bley T, editors. Filaments in Bioprocesses. Springer International Publishing; 2015. pp. 91–132.

57. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25: 955–964. pmid:9023104

58. Sugahara J, Yachie N, Arakawa K, Tomita M. In silico screening of archaeal tRNA-encoding genes having multiple introns with bulge-helix-bulge splicing motifs. RNA. 2007;13: 671–681. pmid:17369313

59. Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004;32: 11–16. pmid:14704338

60. Kinouchi M, Kanaya S, Ikemura T, Kudo Y. Detection of tRNA based on the cloverleaf secondary structure. Genome Inform. 2000;11: 301–302.

61. Sharp S, DeFranco D, Silberklang M, Hosbach HA, Schmidt T, Kubli E, et al. The initiator tRNA genes of Drosophila melanogaster: evidence for a tRNA pseudogene. Nucleic Acids Res. 1981;9: 5867–5882. pmid:6273811

62. Hamada M, Sakulich AL, Koduru SB, Maraia RJ. Transcription termination by RNA polymerase III in fission yeast. A genetic and biochemically tractable model system. J Biol Chem. 2000;275: 29076–29081. pmid:10843998

63. Jühling F, Mörl M, Hartmann RK, Sprinzl M, Stadler PF, Pütz J. tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res. 2009;37: D159–D162. pmid:18957446

64. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, et al. Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28: 1647–1649. pmid:22543367

65. Aslanidis C, Jong PJ de. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 1990;18: 6069–6074. pmid:2235490

66. Bennett JW, Lasure LL. Appendix B—Growth Media. In: Bennett JW, Lasure LL, editors. More Gene Manipulations in Fungi. San Diego: Academic Press; 1991. pp. 441–458.
All items in Spectrum are protected by copyright, with all rights reserved. The use of items is governed by Spectrum's terms of access.

Repository Staff Only: item control page

Downloads per month over past year

Research related to the current document (at the CORE website)
- Research related to the current document (at the CORE website)
Back to top Back to top