The CRISPR toolbox in medical mycology: State of the art and perspectives

Autoři: Florent Morio aff001;  Lisa Lombardi aff001;  Geraldine Butler aff001
Působiště autorů: School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin, Ireland aff001;  Département de Parasitologie et Mycologie Médicale, Université de Nantes, Nantes Université, EA1155 –IICiMed, Nantes, France aff002
Vyšlo v časopise: The CRISPR toolbox in medical mycology: State of the art and perspectives. PLoS Pathog 16(1): e32767. doi:10.1371/journal.ppat.1008201
Kategorie: Review
doi: 10.1371/journal.ppat.1008201


Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR–Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR–Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.

Klíčová slova:

Aspergillus fumigatus – Candida albicans – CRISPR – Cryptococcus neoformans – Fungal genetics – Fungal genomics – Fungal pathogens – Saccharomyces cerevisiae


1. Cole DC, Govender NP, Chakrabarti A, Sacarlal J, Denning DW. Improvement of fungal disease identification and management: combined health systems and public health approaches. Lancet Infect Dis. 2017 Dec;17(12):e412–e419. doi: 10.1016/S1473-3099(17)30308-0 28774694

2. Bongomin F, Gago S, Oladele R, Denning D. Global and Multi-National Prevalence of Fungal Diseases—Estimate Precision. J Fungi. 2017 Oct 18;3(4):57.

3. Chowdhary A, Sharma C, Meis JF. Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog. 2017 May 18;13(5):e1006290. doi: 10.1371/journal.ppat.1006290 28542486

4. Fisher MC, Hawkins NJ, Sanglard D, Gurr SJ. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science. 2018 May 18;360(6390):739–742. doi: 10.1126/science.aap7999 29773744

5. Samaranayake DP, Hanes SD. Milestones in Candida albicans gene manipulation. Fungal Genet Biol. 2011;48(9):858–865. doi: 10.1016/j.fgb.2011.04.003 21511047

6. Defosse TA, Courdavault V, Coste AT, Clastre M, de Bernonville TD, Godon C, et al. A standardized toolkit for genetic engineering of CTG clade yeasts. J Microbiol Methods. 2018 Jan;144:152–156. doi: 10.1016/j.mimet.2017.11.015 29155237

7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816–821 doi: 10.1126/science.1225829 22745249

8. 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 Apr;41(7):4336–4343. doi: 10.1093/nar/gkt135 23460208

9. 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 U S A. 2012 Sep 25;109(39):E2579–2586. doi: 10.1073/pnas.1208507109 22949671

10. Ceccaldi R, Rondinelli B, D’Andrea AD. Repair Pathway Choices and Consequences at the Double-Strand Break. Trends Cell Biol. 2016 Jan;26(1):52–64. doi: 10.1016/j.tcb.2015.07.009 26437586

11. Raschmanová H, Weninger A, Glieder A, Kovar K, Vogl T. Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects. Biotechnol Adv. 2018;36(3):641–665. doi: 10.1016/j.biotechadv.2018.01.006 29331410

12. Caldecott KW. Single-strand break repair and genetic disease. Nat Rev Genet. 2008 Aug;9(8):619–631. doi: 10.1038/nrg2380 18626472

13. Rouet P, Smih F, Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol. 1994 Dec;14(12):8096–8106. doi: 10.1128/mcb.14.12.8096 7969147

14. Smih F, Rouet P, Romanienko PJ, Jasin M. Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res. 1995 Dec 25;23(24):5012–5019. doi: 10.1093/nar/23.24.5012 8559659

15. Storici F, Durham CL, Gordenin DA, Resnick MA. Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):14994–14999. doi: 10.1073/pnas.2036296100 14630945

16. Fuller KK, Chen S, Loros JJ, Dunlap JC. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot Cell. 2015 Nov;14(11):1073–1080. doi: 10.1128/EC.00107-15 26318395

17. Wang Y, Wei D, Zhu X, Pan J, Zhang P, Huo L, et al. A “suicide” CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans. Sci Rep. 2016 Aug 9;6:31145. doi: 10.1038/srep31145 27503169

18. Vyas VK, Barrasa MI, Fink GR. A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci Adv. 2015;1(3):e1500248. doi: 10.1126/sciadv.1500248 25977940

19. Enkler L, Richer D, Marchand AL, Ferrandon D, Jossinet F. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system. Sci Rep. 2016 Dec 21;6(1):35766.

20. Norton EL, Sherwood RK, Bennett RJ. Development of a CRISPR-Cas9 System for Efficient Genome Editing of Candida lusitaniae. mSphere. 2017 Jun 21;2(3). pii: e00217–17. doi: 10.1128/mSphere.00217-17 28657072

21. Ng H, Dean N. Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression. mSphere. 2017 Apr 19;2(2). pii: e00385–16. doi: 10.1128/mSphere.00385-16 28435892

22. Nosek J, Adamíkovâ Ľ, Zemanová J, Tomáška Ľ, Zufferey R, Mamoun C Ben. Genetic manipulation of the pathogenic yeast Candida parapsilosis. Curr Genet. 2002 Oct;42(1):27–35. doi: 10.1007/s00294-002-0326-7 12420143

23. Krappmann S. CRISPR-Cas9, the new kid on the block of fungal molecular biology. Med Mycol. 2017 Jan 1;55(1):16–23. doi: 10.1093/mmy/myw097 27811178

24. Papp T, Csernetics Á, Nyilasi I, ÁBrÓk M, VÁgvÓlgyi C. Genetic Transformation of Zygomycetes Fungi. In: Progress in Mycology. Dordrecht: Springer Netherlands; 2010. p. 75–94.

25. Grahl N, Demers EG, Crocker AW, Hogan DA. Use of RNA-Protein Complexes for Genome Editing in Non- albicans Candida Species. mSphere. 2017 Jun 21;2(3). pii: e00218–17. doi: 10.1128/mSphere.00218-17 28657070

26. Al Abdallah Q, Ge W, Fortwendel JR. A Simple and Universal System for Gene Manipulation in Aspergillus fumigatus: In Vitro-Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates. mSphere. 2018 Jun 13;3(3). pii: e00208–18. doi: 10.1128/mSphereDirect.00208-18

27. Wang P. Two Distinct Approaches for CRISPR-Cas9-Mediated Gene Editing in Cryptococcus neoformans and Related Species. mSphere. 2018;3(3):1–9.

28. Nagy G, Szebenyi C, Csernetics Á, Vaz AG, Tóth EJ, Vágvölgyi C, et al. Development of a plasmid free CRISPR-Cas9 system for the genetic modification of Mucor circinelloides. Sci Rep. 2017 Dec 1;7(1):16800. doi: 10.1038/s41598-017-17118-2 29196656

29. Lombardi L, Oliveira-Pacheco J, Butler G. Plasmid-Based CRISPR-Cas9 Gene Editing in Multiple Candida Species. mSphere. 2019 Feb 13;4(2):557926.

30. Vyas VK, Bushkin GG, Bernstein DA, Getz MA, Sewastianik M, Barrasa MI, et al. New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi. mSphere. 2018 Apr 25;3(2). pii: e00154–18. doi: 10.1128/mSphere.00154-18 29695624

31. Meussen BJ, de Graaff LH, Sanders JPM, Weusthuis RA. Metabolic engineering of Rhizopus oryzae for the production of platform chemicals. Appl Microbiol Biotechnol. 2012 May 13;94(4):875–886. doi: 10.1007/s00253-012-4033-0 22526790

32. Mitchell AP. Location, location, location: Use of CRISPR-Cas9 for genome editing in human pathogenic fungi. PLoS Pathog. 2017 Mar 30;13(3):e1006209. doi: 10.1371/journal.ppat.1006209 28358867

33. Min K, Ichikawa Y, Woolford CA, Mitchell AP. Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System. mSphere. 2017 Mar 15;2(2). pii: e00050–17. doi: 10.1128/mSphere.00050-17

34. Huang MY, Mitchell AP. Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision. mSphere. 2017;2(2):e00050–17. doi: 10.1128/mSphere.00050-17 28317025

35. Nguyen N, Quail MMF, Hernday AD. An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans. mSphere. 2017 Apr 26;2(2). pii: e00149–17. doi: 10.1128/mSphereDirect.00149-17 28497115

36. Shapiro RS, Chavez A, Porter CBM, Hamblin M, Kaas CS, DiCarlo JE, et al. A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nat Microbiol. 2018 Jan;3(1):73–82. doi: 10.1038/s41564-017-0043-0 29062088

37. DiCarlo JE, Chavez A, Dietz SL, Esvelt KM, Church GM. Safeguarding CRISPR-Cas9 gene drives in yeast. Nat Biotechnol. 2015 Dec;33(12):1250–1255. doi: 10.1038/nbt.3412 26571100

38. Hickman MA, Zeng G, Forche A, Hirakawa MP, Abbey D, Harrison BD, et al. The “obligate diploid” Candida albicans forms mating-competent haploids. Nature. 2013;494(7435):55–59. doi: 10.1038/nature11865 23364695

39. Lombardi L, Turner SA, Zhao F, Butler G. Gene editing in clinical isolates of Candida parapsilosis using CRISPR/Cas9. Sci Rep. 2017 Aug 14;7(1):8051. doi: 10.1038/s41598-017-08500-1 28808289

40. Zoppo M, Lombardi L, Rizzato C, Lupetti A, Bottai D, Papp C, et al. CORT0C04210 is required for Candida orthopsilosis adhesion to human buccal cells. Fungal Genet Biol. 2018 Nov;120:19–29. doi: 10.1016/j.fgb.2018.09.001 30205198

41. Morio F, Lombardi L, Binder U, Loge C, Robert E, Graessle D, et al. Precise genome editing using a CRISPR-Cas9 method highlights the role of CoERG11 amino acid substitutions in azole resistance in Candida orthopsilosis. J Antimicrob Chemother. 2019 Aug 1;74(8):2230–2238. doi: 10.1093/jac/dkz204 31106355

42. Cannon RD, Jenkinson HF, Shepherd MG. Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae. Mol Gen Genet. 1990 Apr;221(2):210–218. doi: 10.1007/bf00261723 2196431

43. Schwarzmüller T, Ma B, Hiller E, Istel F, Tscherner M, Brunke S, et al. Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes. PLoS Pathog. 2014 Jun 19;10(6):e1004211. doi: 10.1371/journal.ppat.1004211 24945925

44. Maroc L, Fairhead C. A new inducible CRISPR-Cas9 system useful for genome editing and study of double-strand break repair in Candida glabrata. Yeast. 2019 Aug 18. doi: 10.1002/yea.3440 [Epub ahead of print] 31423617

45. de San Vicente KM, Schröder MS, Lombardi L, Iracane E, Butler G. Correlating Genotype and Phenotype in the Asexual Yeast Candida orthopsilosis Implicates ZCF29 in Sensitivity to Caffeine. G3 (Bethesda). 2019 Sep 4;9(9):3035–3043.

46. Kapoor M, Moloney M, Soltow QA, Pillar CM, Shaw KJ. Evaluation of Resistance Development to the Gwt1 inhibitor Manogepix (APX001A) in Candida Species. Antimicrob Agents Chemother. 2019 Oct 14. pii: AAC.01387-19.

47. Rybak JM, Doorley LA, Nishimoto AT, Barker KS, Palmer GE, Rogers PD. Abrogation of Triazole Resistance upon Deletion of CDR1 in a Clinical Isolate of Candida auris. Antimicrob Agents Chemother. 2019 Mar 27;63(4).

48. Krappmann S, Sasse C, Braus GH. Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end- joining-deficient genetic background. Eukaryot Cell. 2006 Jan;5(1):212–215. doi: 10.1128/EC.5.1.212-215.2006 16400185

49. 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. doi: 10.1016/j.fgb.2015.12.007 26701308

50. Weber J, Valiante V, Nødvig CS, Mattern DJ, Slotkowski RA, Mortensen UH, et al. Functional reconstitution of a fungal natural product gene cluster by advanced genome editing. ACS Synth Biol. 2017 Jan 20;6(1):62–68. doi: 10.1021/acssynbio.6b00203 27611015

51. Umeyama T, Hayashi Y, Shimosaka H, Inukai T, Yamagoe S, Takatsuka S, et al. Cas9/CRISPR genome editing to demonstrate the contribution of Cyp51A Gly138Ser to azole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother. 2018 Aug 27;62(9). pii: e00894–18. doi: 10.1128/AAC.00894-18 29914956

52. Ballard E, Weber J, Melchers WJG, Tammireddy S, Whitfield PD, Brakhage AA, Brown AJP, Verweij PE, Warris A. Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism. Fungal Genet Biol. 2019 Sep;130:98–106. doi: 10.1016/j.fgb.2019.05.005 31128273

53. Hagen F, Khayhan K, Theelen B, Kolecka A, Polacheck I, Sionov E, et al. Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet Biol. 2015 May;78:16–48. doi: 10.1016/j.fgb.2015.02.009 25721988

54. Lin X, Chacko N, Wang L, Pavuluri Y. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation. Med Mycol. 2015 Apr 1;53(3):225–234. doi: 10.1093/mmy/myu083 25541555

55. Davidson RC, Cruz MC, Sia RAL, Allen B, Alspaugh JA, Heitman J. Gene Disruption by Biolistic Transformation in Serotype D Strains of Cryptococcus neoformans. Fungal Genet Biol. 2000 Feb;29(1):38–48. doi: 10.1006/fgbi.1999.1180 10779398

56. Chen Y, Toffaletti DL, Tenor JL, Litvintseva AP, Fang C, Mitchell TG, et al. The Cryptococcus neoformans transcriptome at the site of human meningitis. MBio. 2014 Feb 4;5(1):e01087–13. doi: 10.1128/mBio.01087-13 24496797

57. Goins CL, Gerik KJ, Lodge JK. Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes. Fungal Genet Biol. 2006 Aug;43(8):531–544. doi: 10.1016/j.fgb.2006.02.007 16714127

58. Arras SDM, Chua SMH, Wizrah MSI, Faint JA, Yap AS, Fraser JA. Targeted genome editing via CRISPR in the pathogen Cryptococcus neoformans. PLoS ONE. 2016 Oct 6;11(10):e0164322. doi: 10.1371/journal.pone.0164322 27711143

59. Gao Y, Zhao Y. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol. 2014 Apr;56(4):343–349. doi: 10.1111/jipb.12152 24373158

60. Fan Y, Lin X. Multiple applications of a transient CRISPR-Cas9 coupled with electroporation (TRACE) system in the cryptococcus neoformans species complex. Genetics. 2018 Apr;208(4):1357–1372. doi: 10.1534/genetics.117.300656 29444806

61. Bruni GO, Zhong K, Lee SC, Wang P. CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar.Fungal Genet Biol. 2019 Mar;124:1–7. doi: 10.1016/j.fgb.2018.12.002 30562583

62. Lee KT, So YS, Yang DH, Jung KW, Choi J, Lee DG, Kwon H, Jang J, Wang LL, Cha S, Meyers GL, Jeong E, Jin JH, Lee Y, Hong J, Bang S, Ji JH, Park G, Byun HJ, Park SW, Park YM, Adedoyin G, Kim T, Averette AF, Choi JS, Heitman J, Cheong E, Lee YH, Bahn YS. Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans. Nat Commun. 2016 Sep 28;7:12766. doi: 10.1038/ncomms12766 27677328

63. Noble S. M., French S., Kohn L. A., Chen V. & Johnson A. D. Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity. Nat. Genet. 42, 590–598 (2010). doi: 10.1038/ng.605

64. Holland LM, Schröder MS, Turner SA, Taff H, Andes D, Grózer Z, Gácser A, Ames L, Haynes K, Higgins DG, Butler G. Comparative phenotypic analysis of the major fungal pathogens Candida parapsilosis and Candida albicans. PLoS Pathog. 2014 Sep 18;10(9):e1004365. doi: 10.1371/journal.ppat.1004365 25233198

65. Roemer T, Jiang B, Davison J, Ketela T, Veillette K, Breton A, Tandia F, Linteau A, Sillaots S, Marta C, Martel N, Veronneau S, Lemieux S, Kauffman S, Becker J, Storms R, Boone C, Bussey H. Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery. Mol. Microbiol. 50, 167–181 (2003). doi: 10.1046/j.1365-2958.2003.03697.x 14507372

66. Segal ES, Gritsenko V, Levitan A, Yadav B, Dror N, Steenwyk JL, Silberberg Y, Mielich K, Rokas A, Gow NAR, Kunze R, Sharan R, Berman J. Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans. MBio. 2018 Oct 30;9(5).

67. Glazier VE, Murante T, Koselny K, Murante D, Esqueda M, Wall GA, Wellington M, Hung CY, Kumar A, Krysan DJ. Systematic Complex Haploinsufficiency-Based Genetic Analysis of Candida albicans Transcription Factors: Tools and Applications to Virulence-Associated Phenotypes. G3 (Bethesda). 2018 Mar 28;8(4):1299–1314.

68. Knott GJ, Doudna JA. CRISPR-Cas guides the future of genetic engineering. Science. 2018 Aug 31;361(6405):866–869. doi: 10.1126/science.aat5011 30166482

69. Paquet D, Kwart D, Chen A, Sproul A, Jacob S, Teo S, et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature. 2016 May 27;533(7601):125–129. doi: 10.1038/nature17664 27120160

70. Hao H, Wang X, Jia H, Yu M, Zhang X, Tang H, et al. Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae. Anal Biochem. 2016 Sep 15;509:118–123. doi: 10.1016/j.ab.2016.07.008 27402178

71. Li Z-H, Liu M, Lyu X-M, Wang F-Q, Wei D-Z. CRISPR/Cpf1 facilitated large fragment deletion in Saccharomyces cerevisiae. J Basic Microbiol. 2018 Dec;58(12):1100–1104. doi: 10.1002/jobm.201800195 30198089

72. Shao Y, Lu N, Xue X, Qin Z. Creating functional chromosome fusions in yeast with CRISPR-Cas9. Nat Protoc. 2019 Aug;14(8):2521–2545. doi: 10.1038/s41596-019-0192-0 31300803

73. Yang F, Teoh F, Tan ASM, Cao Y, Pavelka N, Berman J. Aneuploidy Enables Cross-Adaptation to Unrelated Drugs. Mol Biol Evol. 2019 Aug 1;36(8):1768–1782. doi: 10.1093/molbev/msz104 31028698

74. Gorter de Vries AR, Couwenberg LGF, van den Broek M, de la Torre Cortés P, Ter Horst J, Pronk JT, Daran JG. Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast. Nucleic Acids Res. 2019 Feb 20;47(3):1362–1372. doi: 10.1093/nar/gky1216 30517747

75. Ford CB, Funt JM, Abbey D, Issi L, Guiducci C, Martinez DA, Delorey T, Li BY, White TC, Cuomo C, Rao RP, Berman J, Thompson DA, Regev A. The evolution of drug resistance in clinical isolates of Candida albicans. Elife. 2015 Feb 3;4:e00662. doi: 10.7554/eLife.00662 25646566

76. Huang MY, Woolford CA, May G, McManus CJ, Mitchell AP. Circuit diversification in a biofilm regulatory network. PLoS Pathog. 2019 May 22;15(5):e1007787. doi: 10.1371/journal.ppat.1007787 31116789

77. Wijsman M, Swiat MA, Marques WL, Hettinga JK, van den Broek M, de la Torre Cortés P, et al. A toolkit for rapid CRISPR-SpCas9 assisted construction of hexose-transport-deficient Saccharomyces cerevisiae strains. FEMS Yeast Res. 2019 Jan 1;19(1).

78. Burt A. Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc Biol Sci. 2003 May 7;270(1518):921–928. doi: 10.1098/rspb.2002.2319 12803906

79. Sadhu MJ, Bloom JS, Day L, Siegel JJ, Kosuri S, Kruglyak L. Highly parallel genome variant engineering with CRISPR-Cas9. Nat Genet. 2018 Apr;50(4):510–514. doi: 10.1038/s41588-018-0087-y 29632376

80. Adames NR, Gallegos JE, Peccoud J. Yeast genetic interaction screens in the age of CRISPR/Cas. Curr Genet. 2019 Apr;65(2):307–327. doi: 10.1007/s00294-018-0887-8 30255296

81. Halperin SO, Tou CJ, Wong EB, Modavi C, Schaffer D V., Dueber JE. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature. 2018 Aug;560(7717):248–252. doi: 10.1038/s41586-018-0384-8 30069054

82. Adli M. The CRISPR tool kit for genome editing and beyond. Nat Commun. 2018 May 15;9(1):1911. doi: 10.1038/s41467-018-04252-2 29765029

83. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016 Jan 1;351(6268):84–88. doi: 10.1126/science.aad5227 26628643

84. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016 Jan 28;529(7587):490–495. doi: 10.1038/nature16526 26735016

85. Chen JS, Dagdas YS, Kleinstiver benjamin P, Welch MM, Sousa AA, Harrington LB, et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature. 2017 Oct 19;550(7676):407–410. doi: 10.1038/nature24268 28931002

86. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, et al. Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Oct 22;163(3):759–771. doi: 10.1016/j.cell.2015.09.038 26422227

87. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018 Apr 5;556(7699):57–63. doi: 10.1038/nature26155 29512652

88. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016 May 20;533(7603):420–424. doi: 10.1038/nature17946 27096365

89. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of T to G C in genomic DNA without DNA cleavage. Nature. 2017 Oct 25;551(7681):464–471. doi: 10.1038/nature24644 29160308

90. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019 Oct 21. doi: 10.1038/s41586-019-1711-4 31634902

91. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, et al. RNA targeting with CRISPR-Cas13. Nature. 2017 Oct 12;550(7675):280–284. doi: 10.1038/nature24049 28976959

92. Giersch RM, Finnigan GC. Yeast still a beast: Diverse applications of CRISPR/CAS editing technology in S. cerevisiae. Yale J Biol Med. 2017 Dec 19;90(4):643–651. 29259528

93. Knight SC, Tjian R, Doudna JA. Genomes in Focus: Development and Applications of CRISPR-Cas9 Imaging Technologies. Angew Chem Int Ed Engl. 2018 Apr 9;57(16):4329–4337. doi: 10.1002/anie.201709201 29080263

94. Schultz C, Lian J, Zhao H. Metabolic Engineering of Saccharomyces cerevisiae Using a Trifunctional CRISPR/Cas System for Simultaneous Gene Activation, Interference, and Deletion. Methods Enzymol. 2018;608:265–276. doi: 10.1016/bs.mie.2018.04.010 30173764

95. Wensing L, Sharma J, Uthayakumar D, Proteau Y, Chavez A, Shapiro RS. A CRISPR Interference Platform for Efficient Genetic Repression in Candida albicans. mSphere. 2019 Feb 13;4(1). pii: e00002–19. doi: 10.1128/mSphere.00002-19 30760609

96. Román E, Coman I, Prieto D, Alonso-Monge R, Pla J. Implementation of a CRISPR-Based System for Gene Regulation in Candida albicans. mSphere. 2019 Feb 13;4(1). pii: e00001–19. doi: 10.1128/mSphere.00001-19 30760608

97. Halder V, Porter CBM, Chavez A, Shapiro RS. Design, execution, and analysis of CRISPR-Cas9-based deletions and genetic interaction networks in the fungal pathogen Candida albicans. Nat Protoc. 2019 Mar;14(3):955–975. doi: 10.1038/s41596-018-0122-6 30737491

98. Scorzoni L, de Paula E Silva AC, Marcos CM, Assato PA, de Melo WC, de Oliveira HC, Costa-Orlandi CB, Mendes-Giannini MJ, Fusco-Almeida AM. Antifungal Therapy: New Advances in the Understanding and Treatment of Mycosis. Front Microbiol. 2017 Jan 23;8:36. doi: 10.3389/fmicb.2017.00036 28167935

99. Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan AL, Paul LM, Parham LA, Garcia KF, Barnes KG, Chak B, Mondini A, Nogueira ML, Isern S, Michael SF, Lorenzana I, Yozwiak NL, MacInnis BL, Bosch I, Gehrke L, Zhang F, Sabeti PC.Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018 Apr 27;360(6387):444–448. doi: 10.1126/science.aas8836 29700266

100. Arastehfar A, Wickes BL, Ilkit M, Pincus DH, Daneshnia F, Pan W, Fang W, Boekhout T.Identification of Mycoses in Developing Countries.J Fungi (Basel). 2019 Sep 29;5(4).

101. Quan J, Langelier C, Kuchta A, Batson J, Teyssier N, Lyden A, Caldera S, McGeever A, Dimitrov B, King R, Wilheim J, Murphy M, Ares LP, Travisano KA, Sit R, Amato R, Mumbengegwi DR, Smith JL, Bennett A, Gosling R, Mourani PM, Calfee CS, Neff NF, Chow ED, Kim PS, Greenhouse B, DeRisi JL, Crawford ED.FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences.Nucleic Acids Res. 2019 Aug 22;47(14):e83. doi: 10.1093/nar/gkz418 31114866

102. Bongomin F, Gago S, Oladele RO, Denning DW. Global and Multi-National Prevalence of Fungal Diseases-Estimate Precision. J Fungi (Basel). 2017 Oct 18;3(4).

103. Ianiri G, Dagotto G, Sun S, Heitman J.Advancing Functional Genetics Through Agrobacterium-Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast Malassezia. Genetics. 2019 Aug;212(4):1163–1179. doi: 10.1534/genetics.119.302329 31243056

104. Wang Q, Coleman JJ.CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. Fungal Genet Biol. 2019 May;126:17–24. doi: 10.1016/j.fgb.2019.02.002 30738140

105. Kujoth GC, Sullivan TD, Merkhofer R, Lee TJ, Wang H, Brandhorst T, Wüthrich M, Klein BS. CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis. MBio. 2018 Apr 3;9(2). pii: e00412–18. doi: 10.1128/mBio.00412-18 29615501

106. Sanglard D. Finding the needle in a haystack: Mapping antifungal drug resistance in fungal pathogen by genomic approaches. Hogan DA, editor. PLoS Pathog. 2019 Jan 31;15(1):e1007478. doi: 10.1371/journal.ppat.1007478 30703166

Hygiena a epidemiologie Infekční lékařství Laboratoř

Článek vyšel v časopise

PLOS Pathogens

2020 Číslo 1

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…

Kurzy Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se

VIRTUÁLNÍ ČEKÁRNA ČR Jste praktický lékař nebo pediatr? Zapojte se! Jste praktik nebo pediatr? Zapojte se!