CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms

Abstract

Genetic manipulation of microorganisms has been crucial in understanding their biology, yet for many microbial species, robust tools for comprehensive genetic analysis were lacking until the advent of CRISPR–Cas-based gene editing techniques. In this Progress article, we discuss advances in CRISPR-based techniques for the genetic analysis of genetically intractable microorganisms, with an emphasis on mycobacteria, fungi and parasites. We discuss how CRISPR-based analyses in these organisms have enabled the discovery of novel gene functions, the investigation of genetic interaction networks and the identification of virulence factors.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Applications of different CRISPR technologies in diverse microorganisms.
Fig. 2: Future applications of CRISPR–Cas-based gene editing techniques.

References

  1. 1.

    Griffith, F. The significance of pneumococcal types. J. Hyg. 27, 113 (1928).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Avery, O. T., Macleod, C. M. & McCarty, M. Studies on the chemical nature of the substance inducing transformation of pneumococcal types: induction of transformation by a desoxyribonucleic acid fraction isolated from Pneumococcus type III. J. Exp. Med. 79, 137–158 (1944).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Cohen, S. N., Chang, A. C., Boyer, H. W. & Helling, R. B. Construction of biologically functional bacterial plasmids in vitro. Proc. Natl Acad. Sci. USA 70, 3240–3244 (1973).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391 (2002).

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Costanzo, M. et al. A global genetic interaction network maps a wiring diagram of cellular function. Science 353, aaf1420 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    van Opijnen, T. & Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat. Rev. Microbiol. 11, 435–442 (2013).

    Article  PubMed  Google Scholar 

  8. 8.

    Langridge, G. C. et al. Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants. Genome Res. 19, 2308–2316 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Gallagher, L. A., Shendure, J. & Manoil, C. Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. mBio 2, e00315–10 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Sander, J. D. & Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    DiCarlo, J. E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14.

    Bikard, D. et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat. Biotechnol. 32, 1146–1150 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Citorik, R. J., Mimee, M. & Lu, T. K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat. Biotechnol. 32, 1141–1145 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Dominguez, A. A., Lim, W. A. & Qi, L. S. Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol. 17, 5–15 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    La Russa, M. F. & Qi, L. S. The new state of the art: Cas9 for gene activation and repression. Mol. Cell. Biol. 35, 3800–3809 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Kendall, S. L. & Frita, R. Construction of targeted mycobacterial mutants by homologous recombination. Methods Mol. Biol. 465, 297–310 (2009).

    Article  PubMed  Google Scholar 

  20. 20.

    Choudhary, E., Thakur, P., Pareek, M. & Agarwal, N. Gene silencing by CRISPR interference in mycobacteria. Nat. Commun. 6, 6267 (2015).

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    da Silva Ferreira, M. E. et al. The akuBKU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5, 207–211 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Weld, R. J., Plummer, K. M., Carpenter, M. A. & Ridgway, H. J. Approaches to functional genomics in filamentous fungi. Cell Res. 16, 31–44 (2006).

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Jiang, D. et al. Molecular tools for functional genomics in filamentous fungi: recent advances and new strategies. Biotechnol. Adv. 31, 1562–1574 (2013).

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Peng, D., Kurup, S. P., Yao, P. Y., Minning, T. A. & Tarleton, R. L. CRISPR-Cas9-mediated single-gene and gene family disruption in Trypanosoma cruzi. mBio 6, e02097–14 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Xu, D., Brandán, C. P., Basombrío, M. A. & Tarleton, R. L. Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi. BMC Microbiol. 9, 90 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Meissner, M., Breinich, M. S., Gilson, P. R. & Crabb, B. S. Molecular genetic tools in Toxoplasma and Plasmodium: achievements and future needs. Curr. Opin. Microbiol. 10, 349–356 (2007).

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Donald, R. G. & Roos, D. S. Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in. Toxoplasma gondii. Mol. Biochem. Parasitol. 63, 243–253 (1994).

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Fuller, K. K., Chen, S., Loros, J. J. & Dunlap, J. C. Development of the CRISPR/Cas9 system for targeted gene disruption in. Aspergillus fumigatus. Eukaryot. Cell 14, 1073–1080 (2015).

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Enkler, L., Richer, D., Marchand, A. L., Ferrandon, D. & Jossinet, F. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system. Sci. Rep 6, 35766 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Liu, Q. 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 10, 1 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    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. 1, 15007 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165, 1493–1506 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Singh, A. K. et al. Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system. Nucleic Acids Res. 44, e143 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Rock, J. M. et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat. Microbiol. 2, 16274 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Vyas, V. K., Barrasa, M. I. & Fink, G. R. A. Candida albicans CRISPR system permits genetic engineering of essential genes and gene families. Sci. Adv. 1, e1500248 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Min, K., Ichikawa, Y., Woolford, C. A. & Mitchell, A. P. Candida albicans gene deletion with a transient CRISPR-Cas9 system. mSphere 1, e00130–16 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Grahl, N., Demers, E. G., Crocker, A. W. & Hogan, D. A. Use of RNA-protein complexes for genome editing in non-albicans Candida species. mSphere 2, e00218–17 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Ren, B. & Gupta, N. Taming parasites by tailoring them. Front. Cell. Infect. Microbiol. 7, 292 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Sidik, S. M. et al. A genome-wide CRISPR screen in Toxoplasma identifies essential Apicomplexan genes. Cell 166, 1423–1435.e12 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Ghorbal, M. et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat. Biotechnol. 32, 819–821 (2014).

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Wagner, J. C., Platt, R. J., Goldfless, S. J., Zhang, F. & Niles, J. C. Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum. Nat. Methods 11, 915–918 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Vinayak, S. et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature 523, 477–480 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Zhang, W.-W. & Matlashewski, G. CRISPR-Cas9-mediated genome editing in Leishmania donovani. mBio 6, e00861 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Babu, M. et al. Quantitative genome-wide genetic interaction screens reveal global epistatic relationships of protein complexes in Escherichia coli. PLoS Genet. 10, e1004120 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Butler, G. et al. Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459, 657–662 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Ohtani, N., Tomita, M. & Itaya, M. An extreme thermophile. Thermus thermophilus, is a polyploid bacterium. J. Bacteriol. 192, 5499–5505 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Soppa, J. Polyploidy in archaea and bacteria: about desiccation resistance, giant cell size, long-term survival, enforcement by a eukaryotic host and additional aspects. J. Mol. Microbiol. Biotechnol. 24, 409–419 (2014).

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Dave, K. et al. in Genetic Transformation Systems in Fungi Vol. 2 (eds van den Berg, M. A. & Maruthachalam, K.) 141–153 (Springer, Cham, 2014).

  49. 49.

    Nødvig, C. S., Nielsen, J. B., Kogle, M. E. & Mortensen, U. H. A. CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS ONE 10, e0133085 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Striepen, B. & Soldati, D. In Toxoplasma gondii 1st edn (eds Weiss, L. M. & Kim, K.) 391–418 (Academic Press, 2007).

  51. 51.

    Kangussu-Marcolino, M. M., Cunha, A. P., Avila, A. R., Herman, J.-P. & DaRocha, W. D. Conditional removal of selectable markers in Trypanosoma cruzi using a site-specific recombination tool: proof of concept. Mol. Biochem. Parasitol. 198, 71–74 (2014).

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Shapiro, R. S. et al. A CRISPR-Cas9-based gene drive platform for genetic interaction analysis in Candida albicans. Nat. Microbiol. 3, 73–82 (2018).

    CAS  Article  PubMed  Google Scholar 

  53. 53.

    Norris, A. D., Gracida, X. & Calarco, J. A. CRISPR-mediated genetic interaction profiling identifies RNA binding proteins controlling metazoan fitness. eLife 6, e28129 (2017).

    PubMed  PubMed Central  Google Scholar 

  54. 54.

    Gaj, T., Gersbach, C. A. & Barbas, C. F. 3rd ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Huang, H. et al. CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an autotrophic gas-fermenting bacterium. ACS Synth. Biol. 5, 1355–1361 (2016).

    CAS  Article  PubMed  Google Scholar 

  56. 56.

    Wang, Y. et al. Bacterial genome editing with CRISPR-Cas9: deletion, integration, single nucleotide modification, and desirable ‘clean’ mutant selection in Clostridium beijerinckii as an example. ACS Synth. Biol. 5, 721–732 (2016).

    CAS  Article  PubMed  Google Scholar 

  57. 57.

    Nagaraju, S., Davies, N. K., Walker, D. J. F., Köpke, M. & Simpson, S. D. Genome editing of Clostridium autoethanogenum using CRISPR/Cas9. Biotechnol. Biofuels 9, 219 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Cobb, R. E., Wang, Y. & Zhao, H. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723–728 (2015).

    CAS  Article  PubMed  Google Scholar 

  59. 59.

    Zhang, M. M. et al. CRISPR–Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters. Nat. Chem. Biol. 13, 607–609 (2017).

    CAS  Article  Google Scholar 

  60. 60.

    Pohl, C., Kiel, J. A. K. W., Driessen, A. J. M., Bovenberg, R. A. L. & Nygård, Y. CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synth. Biol. 5, 754–764 (2016).

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    Weninger, A., Hatzl, A.-M., Schmid, C., Vogl, T. & Glieder, A. Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J. Biotechnol. 235, 139–149 (2016).

    CAS  Article  PubMed  Google Scholar 

  62. 62.

    Schwartz, C., Shabbir-Hussain, M., Frogue, K., Blenner, M. & Wheeldon, I. Standardized markerless gene integration for pathway engineering in Yarrowia lipolytica. ACS Synth. Biol. 6, 402–409 (2017).

    CAS  Article  PubMed  Google Scholar 

  63. 63.

    Mohr, S. E., Smith, J. A., Shamu, C. E., Neumüller, R. A. & Perrimon, N. RNAi screening comes of age: improved techniques and complementary approaches. Nat. Rev. Mol. Cell Biol. 15, 591–600 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Shabalina, S. A. & Koonin, E. V. Origins and evolution of eukaryotic RNA interference. Trends Ecol. Evol. 23, 578–587 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  65. 65.

    Rusk, N. Microbiology: Prokaryotic RNAi. Nat. Methods 9, 220–221 (2012).

    CAS  Article  PubMed  Google Scholar 

  66. 66.

    van der Oost, J., Swarts, D. C. & Jore, M. M. Prokaryotic Argonautes — variations on the RNA interference theme. Microb. Cell Fact. 1, 158–159 (2014).

    Article  Google Scholar 

  67. 67.

    Kolev, N. G., Tschudi, C. & Ullu, E. RNA interference in protozoan parasites: achievements and challenges. Eukaryot. Cell 10, 1156–1163 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  68. 68.

    Baum, J. et al. Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites. Nucleic Acids Res. 37, 3788–3798 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  69. 69.

    van Opijnen, T., Bodi, K. L. & Camilli, A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat. Methods 6, 767–772 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  70. 70.

    Li, T. et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res. 39, 6315–6325 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  71. 71.

    Straimer, J. et al. Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases. Nat. Methods 9, 993–998 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Ji, W. et al. Specific gene repression by CRISPRi system transferred through bacterial conjugation. ACS Synth. Biol. 3, 929–931 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  73. 73.

    Yosef, I., Manor, M., Kiro, R. & Qimron, U. Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc. Natl Acad. Sci. USA 112, 7267–7272 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  74. 74.

    Soares Medeiros, L. C. et al. Rapid, selection-free, high-efficiency genome editing in protozoan parasites using CRISPR-Cas9 ribonucleoproteins. mBio 8, e01788–17 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  75. 75.

    Pyne, M. E., Bruder, M. R., Moo-Young, M., Chung, D. A. & Chou, C. P. Harnessing heterologous and endogenous CRISPR-Cas machineries for efficient markerless genome editing in Clostridium. Sci. Rep. 6, 25666 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  76. 76.

    Li, Y. et al. Harnessing Type I and Type III CRISPR-Cas systems for genome editing. Nucleic Acids Res. 44, e34 (2016).

    Article  PubMed  Google Scholar 

  77. 77.

    Schaefer, K. A. et al. Unexpected mutations after CRISPR–Cas9 editing in vivo. Nat. Methods 14, 547–548 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  78. 78.

    Pawluk, A., Davidson, A. R. & Maxwell, K. L. Anti-CRISPR: discovery, mechanism and function. Nat. Rev. Microbiol. 16, 12–17 (2018).

    CAS  Article  PubMed  Google Scholar 

  79. 79.

    Gaudelli, N. M. et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  80. 80.

    Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  81. 81.

    Kuscu, C. et al. CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations. Nat. Methods 14, 710–712 (2017).

    CAS  Article  PubMed  Google Scholar 

  82. 82.

    Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  83. 83.

    Luesch, H. et al. A genome-wide overexpression screen in yeast for small-molecule target identification. Chem. Biol. 12, 55–63 (2005).

    CAS  Article  PubMed  Google Scholar 

  84. 84.

    Kitagawa, M. et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 12, 291–299 (2005).

    CAS  Article  PubMed  Google Scholar 

  85. 85.

    Liu, X. S. et al. Editing DNA methylation in the mammalian genome. Cell 167, 233–247.e17 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  86. 86.

    Cohen, N. R. et al. A role for the bacterial GATC methylome in antibiotic stress survival. Nat. Genet. 48, 581–586 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Rai, L. S., Singha, R., Brahma, P. & Sanyal, K. Epigenetic determinants of phenotypic plasticity in. Candida albicans. Fungal Biol. Rev. 32, 10–19 (2018).

    Article  Google Scholar 

  88. 88.

    Robert McMaster, W., Morrison, C. J. & Kobor, M. S. Epigenetics: a new model for intracellular parasite–host cell regulation. Trends Parasitol. 32, 515–521 (2016).

    CAS  Article  PubMed  Google Scholar 

  89. 89.

    Adamson, B. et al. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell 167, 1867–1882.e21 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  91. 91.

    Kanjee, U. et al. CRISPR/Cas9 knockouts reveal genetic interaction between strain-transcendent erythrocyte determinants of Plasmodium falciparum invasion. Proc. Natl Acad. Sci. USA 114, E9356–E9365 (2017).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  92. 92.

    Friedland, A. E. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741–743 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  93. 93.

    Hwang, W. Y. et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  94. 94.

    Li, J.-F. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat. Biotechnol. 31, 688–691 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Work in the authors’ laboratory was supported by the Defense Threat Reduction Agency grant HDTRA1-15-1-0051, the Paul G. Allen Frontiers Group, the Wyss Institute for Biologically Inspired Engineering and the Broad Institute of MIT and Harvard. A.C. acknowledges support from the Burroughs Wellcome Fund Career Award for Medical Scientists.

Author information

Affiliations

Authors

Contributions

R.S.S. researched data for the article and wrote the article. J.J.C., A.C. and R.S.S. made substantial contributions to discussions of the content and reviewed and edited the manuscript before submission.

Corresponding author

Correspondence to James J. Collins.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shapiro, R.S., Chavez, A. & Collins, J.J. CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms. Nat Rev Microbiol 16, 333–339 (2018). https://doi.org/10.1038/s41579-018-0002-7

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing