The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development

  • Max S. FarnworthEmail author
  • Kolja N. Eckermann
  • Hassan M. M. Ahmed
  • Dominik S. Mühlen
  • Bicheng He
  • Gregor BucherEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)


With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.


CRISPR/Cas9 Genome engineering HDR NHEJ Tribolium Neural lineage Brain development 2A peptide 



We express our gratitude to Prof. Martin Klingler for discussions on the gene-specific enhancer trap strategy and Dr. Stefan Dippel for discussions on the bicistronic line strategy. In addition, we want to thank Patricio Ferrer Murguia for useful additional information.


  1. 1.
    Perry M, Konstantinides N, Pinto-Teixeira F, Desplan C (2017) Generation and evolution of neural cell types and circuits: insights from the Drosophila visual system. Annu Rev Genet 51:501–527CrossRefGoogle Scholar
  2. 2.
    Doe CQ (2017) Temporal patterning in the Drosophila CNS. Annu Rev Cell Dev Biol 33:219–240CrossRefGoogle Scholar
  3. 3.
    Urbach R, Technau GM (2004) Neuroblast formation and patterning during early brain development in Drosophila. BioEssays 26:739–751CrossRefGoogle Scholar
  4. 4.
    Hartenstein V, Stollewerk A (2015) The evolution of early neurogenesis. Dev Cell 32:390–407CrossRefGoogle Scholar
  5. 5.
    Arendt D, Tosches MA, Marlow H (2016) From nerve net to nerve ring, nerve cord and brain—evolution of the nervous system. Nat Rev Neurosci 17:61–72CrossRefGoogle Scholar
  6. 6.
    El Jundi B, Heinze S Three-dimensional atlases of insect brains. Neurohistology and Imaging: Basic TechniquesGoogle Scholar
  7. 7.
    Koniszewski NDB, Kollmann M, Bigham M, Farnworth M, He B, Büscher M, Hütteroth W, Binzer M, Schachtner J, Bucher G (2016) The insect central complex as model for heterochronic brain development—background, concepts, and tools. Dev Genes Evol 226:209–219CrossRefGoogle Scholar
  8. 8.
    Brown SJ, Mahaffey JP, Lorenzen MD, Denell RE, Mahaffey JW (1999) Using RNAi to investigate orthologous homeotic gene function during development of distantly related insects. Evol Dev 1:11–15CrossRefGoogle Scholar
  9. 9.
    Bucher G, Scholten J, Klingler M (2002) Parental RNAi in Tribolium (Coleoptera). Curr Biol 12:R85–R86CrossRefGoogle Scholar
  10. 10.
    Tomoyasu Y, Denell RE (2004) Larval RNAi in Tribolium (Coleoptera) for analyzing adult development. Dev Genes Evol 214:575–578CrossRefGoogle Scholar
  11. 11.
    Schmitt-Engel C, Schultheis D, Schwirz J et al (2015) The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun 6:7822CrossRefGoogle Scholar
  12. 12.
    Berghammer AJ, Klingler M, Wimmer EA (1999) Genetic techniques: A universal marker for transgenic insects. Nature 402:370–371CrossRefGoogle Scholar
  13. 13.
    Trauner J, Schinko J, Lorenzen MD, Shippy TD, Wimmer EA, Beeman RW, Klingler M, Bucher G, Brown SJ (2009) Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps. BMC Biol 7:73CrossRefGoogle Scholar
  14. 14.
    Lorenzen MD, Kimzey T, Shippy TD, Brown SJ, Denell RE, Beeman RW (2007) piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids. Insect Mol Biol 16:265–275CrossRefGoogle Scholar
  15. 15.
    Gilles AF, Schinko JB, Averof M (2015) Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum. Development 142:2832–2839CrossRefGoogle Scholar
  16. 16.
    Gilles AF, Schinko JB, Schacht MI, Enjolras C, Averof M (2019) Clonal analysis by tunable CRISPR-mediated excision. Development 146 (1):dev170969CrossRefGoogle Scholar
  17. 17.
    Hayashi S, Ito K, Sado Y et al (2002) GETDB, a database compiling expression patterns and molecular locations of a collection of gal4 enhancer traps. Genesis 34:58–61CrossRefGoogle Scholar
  18. 18.
    Mollereau B, Wernet MF, Beaufils P, Killian D, Pichaud F, Kühnlein R, Desplan C (2000) A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells. Mech Dev 93:151–160CrossRefGoogle Scholar
  19. 19.
    O’Kane CJ, Gehring WJ (1987) Detection in situ of genomic regulatory elements in Drosophila. PNAS 84:9123–9127CrossRefGoogle Scholar
  20. 20.
    Pfeiffer BD, Jenett A, Hammonds AS et al (2008) Tools for neuroanatomy and neurogenetics in Drosophila. Proc Natl Acad Sci U S A 105:9715–9720CrossRefGoogle Scholar
  21. 21.
    Wu JS, Luo L (2006) A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining. Nat Protoc 1:2110–2115CrossRefGoogle Scholar
  22. 22.
    Jin EJ, Kiral FR, Ozel MN, Burchardt LS, Osterland M, Epstein D, Wolfenberg H, Prohaska S, Hiesinger PR (2018) Live Observation of Two Parallel Membrane Degradation Pathways at Axon Terminals. Curr Biol 28:1027–1038.e4CrossRefGoogle Scholar
  23. 23.
    Eckert C, Aranda M, Wolff C, Tautz D (2004) Separable stripe enhancer elements for the pair-rule gene hairy in the beetle Tribolium. EMBO Rep 5:638–642CrossRefGoogle Scholar
  24. 24.
    Wilson C, Bellen H, Gehring W (1990) Position Effects on Eukaryotic Gene-Expression. Annu Rev Cell Biol 6:679–714CrossRefGoogle Scholar
  25. 25.
    John AV, Sramkoski LL, Walker EA, Cooley AM, Wittkopp PJ (2016) Sensitivity of allelic divergence to genomic position: Lessons from the Drosophila tan gene. G3 6:2955–2962CrossRefGoogle Scholar
  26. 26.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A Programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821CrossRefGoogle Scholar
  27. 27.
    Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278CrossRefGoogle Scholar
  28. 28.
    Gratz SJ, Wildonger J, Harrison MM, O’Connor-Giles KM (2013) CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly 7:249–255CrossRefGoogle Scholar
  29. 29.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096CrossRefGoogle Scholar
  30. 30.
    Rylee JC, Siniard DJ, Doucette K, Zentner GE, Zelhof AC (2018) Expanding the genetic toolkit of Tribolium castaneum. PLoS One 13:e0195977CrossRefGoogle Scholar
  31. 31.
    Dönitz J, Schmitt-Engel C, Grossmann D, Gerischer L, Tech M, Schoppmeier M, Klingler M, Bucher G (2015) iBeetle-Base: a database for RNAi phenotypes in the red flour beetle Tribolium castaneum. Nucl Acids Res 43:D720–D725CrossRefGoogle Scholar
  32. 32.
    Dönitz J, Gerischer L, Hahnke S, Pfeiffer S, Bucher G (2018) Expanded and updated data and a query pipeline for iBeetle-Base. Nucleic Acids Res 46:D831–D835CrossRefGoogle Scholar
  33. 33.
    Lai Y-T, Deem KD, Borràs-Castells F, Sambrani N, Rudolf H, Suryamohan K, El-Sherif E, Halfon MS, McKay DJ, Tomoyasu Y (2018) Enhancer identification and activity evaluation in the red flour beetle, Tribolium castaneum. Development. Scholar
  34. 34.
    Häcker U, Nystedt S, Barmchi MP, Horn C, Wimmer EA (2003) piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila. PNAS 100:7720–7725CrossRefGoogle Scholar
  35. 35.
    Kvon EZ, Kazmar T, Stampfel G, Yáñez-Cuna JO, Pagani M, Schernhuber K, Dickson BJ, Stark A (2014) Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature 512:91–95CrossRefGoogle Scholar
  36. 36.
    Schinko JB, Weber M, Viktorinova I, Kiupakis A, Averof M, Klingler M, Wimmer EA, Bucher G (2010) Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters. BMC Dev Biol 10:53CrossRefGoogle Scholar
  37. 37.
    Schinko JB, Hillebrand K, Bucher G (2012) Heat shock-mediated misexpression of genes in the beetle Tribolium castaneum. Dev Genes Evol 222:287–298CrossRefGoogle Scholar
  38. 38.
    Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479CrossRefGoogle Scholar
  39. 39.
    Lorenzen MD, Brown SJ, Denell RE, Beeman RW (2002) Cloning and characterization of the Tribolium castaneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase. Genetics 160:225–234PubMedPubMedCentralGoogle Scholar
  40. 40.
    Sarov M, Barz C, Jambor H et al (2016) A genome-wide resource for the analysis of protein localisation in Drosophila. elife 5:e12068CrossRefGoogle Scholar
  41. 41.
    Donnelly MLL, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 82:1013–1025CrossRefGoogle Scholar
  42. 42.
    Szymczak-Workman AL, Vignali KM, Vignali DAA (2012) Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc 2012. pdb.ip067876-pdb.ip067876Google Scholar
  43. 43.
    Kim JH, Lee S-R, Li L-H, Park H-J, Park J-H, Lee KY, Kim M-K, Shin BA, Choi S-Y (2011) High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6:e18556CrossRefGoogle Scholar
  44. 44.
    Brown SJ, Shippy TD, Miller S, Bolognesi R, Beeman RW, Lorenzen MD, Bucher G, Wimmer EA, Klingler M (2009) The red flour beetle, Tribolium castaneum (Coleoptera): a model for studies of development and pest biology. Cold Spring Harb Protoc 2009:pdb.emo126CrossRefGoogle Scholar
  45. 45.
    Tribolium Genome Sequencing Consortium, Richards S, Gibbs RA, et al (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452:949–955Google Scholar
  46. 46.
    Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196:961–971CrossRefGoogle Scholar
  47. 47.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefGoogle Scholar
  48. 48.
    Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832CrossRefGoogle Scholar
  49. 49.
    Horn C, Schmid BGM, Pogoda FS, Wimmer EA (2002) Fluorescent transformation markers for insect transgenesis. Insect Biochem Mol Biol 32:1221–1235CrossRefGoogle Scholar
  50. 50.
    Ulrich A, Andersen KR, Schwartz TU (2012) Exponential megapriming PCR (EMP) cloning—seamless DNA insertion into any target plasmid without sequence constraints. PLoS One 7:e53360CrossRefGoogle Scholar
  51. 51.
    Beumer KJ, Trautman JK, Mukherjee K, Carroll D (2013) Donor DNA utilization during gene targeting with zinc-finger nucleases. G3 3:657–664Google Scholar
  52. 52.
    Tycko J, Myer VE, Hsu PD (2016) Methods for optimizing CRISPR-Cas9 genome editing specificity. Mol Cell 63:355–370CrossRefGoogle Scholar
  53. 53.
    Berghammer A, Bucher G, Maderspacher F, Klingler M (1999) A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum. Dev Genes Evol 209:382–389CrossRefGoogle Scholar
  54. 54.
    Posnien N, Schinko J, Grossmann D, Shippy TD, Konopova B, Bucher G (2009) RNAi in the red flour beetle (Tribolium). Cold Spring Harb Protoc 2009:pdb.prot5256CrossRefGoogle Scholar
  55. 55.
    Eckermann KN, Ahmed HMM, KaramiNejadRanjbar M, Dippel S, Ogaugwu CE, Kitzmann P, Isah MD, Wimmer EA (2018) Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species. Insect Biochem Mol Biol 98:16–24CrossRefGoogle Scholar
  56. 56.
    Strobl F, Ross JA, Stelzer EHK (2017) Non-lethal genotyping of Tribolium castaneum adults using genomic DNA extracted from wing tissue. PLoS One 12:e0182564CrossRefGoogle Scholar
  57. 57.
    Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 is a single RNA-guided endonuclease of a Class 2 CRISPR-Cas system. Cell 163:759–771CrossRefGoogle Scholar
  58. 58.
    Labun K, Montague TG, Gagnon JA, Thyme SB, Valen E (2016) CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44:W272–W276CrossRefGoogle Scholar
  59. 59.
    Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407CrossRefGoogle Scholar
  60. 60.
    Clarke R, Heler R, MacDougall MS, Yeo NC, Chavez A, Regan M, Hanakahi L, Church GM, Marraffini LA, Merrill BJ (2018) Enhanced bacterial immunity and mammalian genome editing via rna-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks. Mol Cell 71:42–55. e8CrossRefGoogle Scholar
  61. 61.
    Port F, Chen H-M, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci 111:E2967–E2976CrossRefGoogle Scholar
  62. 62.
    Bassett AR, Tibbit C, Ponting CP, Liu J-L (2013) Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep 4:220–228CrossRefGoogle Scholar
  63. 63.
    Keeler KJ, Dray T, Penney JE, Gloor GB (1996) Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster. Mol Cell Biol 16:522–528CrossRefGoogle Scholar
  64. 64.
    Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, Maeder ML, Joung JK, Chen Z-Y, Liu DR (2015) Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33:73–80CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Max S. Farnworth
    • 1
    • 2
    Email author
  • Kolja N. Eckermann
    • 2
    • 3
  • Hassan M. M. Ahmed
    • 3
    • 4
  • Dominik S. Mühlen
    • 1
    • 2
  • Bicheng He
    • 1
  • Gregor Bucher
    • 1
    Email author
  1. 1.Department of Evolutionary Developmental Genetics, Johann-Friedrich-Blumenbach InstituteGZMB, University of GöttingenGöttingenGermany
  2. 2.Göttingen Graduate Center for Molecular Biosciences, Neurosciences and BiophysicsGöttingenGermany
  3. 3.Department of Developmental Biology, Johann-Friedrich-Blumenbach InstituteGZMB, University of GöttingenGöttingenGermany
  4. 4.Department of Crop Protection, Faculty of AgricultureUniversity of Khartoum, Khartoum-NorthKhartoumSudan

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