Abstract
The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.
Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Herm-Gotz A, Agop-Nersesian C, Munter S, Grimley JS, Wandless TJ, Frischknecht F, Meissner M (2007) Rapid control of protein level in the apicomplexan Toxoplasma gondii. Nat Methods 4(12):1003–1005. https://doi.org/10.1038/nmeth1134
Andenmatten N, Egarter S, Jackson AJ, Jullien N, Herman JP, Meissner M (2013) Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat Methods 10(2):125–127. https://doi.org/10.1038/nmeth.2301
Pieperhoff MS, Pall GS, Jimenez-Ruiz E, Das S, Melatti C, Gow M, Wong EH, Heng J, Muller S, Blackman MJ, Meissner M (2015) Conditional U1 gene silencing in Toxoplasma gondii. PLoS One 10(6):e0130356. https://doi.org/10.1371/journal.pone.0130356
Long S, Brown KM, Drewry LL, Anthony B, Phan IQH, Sibley LD (2017) Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii. PLoS Pathog 13(5):e1006379. https://doi.org/10.1371/journal.ppat.1006379
Meissner M, Brecht S, Bujard H, Soldati D (2001) Modulation of myosin a expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii. Nucleic Acids Res 29(22):E115
Meissner M, Schluter D, Soldati D (2002) Role of Toxoplasma gondii myosin a in powering parasite gliding and host cell invasion. Science 298(5594):837–840. https://doi.org/10.1126/science.1074553
Brown KM, Long S, Sibley LD (2017) Plasma membrane association by N-acylation governs pkg function in Toxoplasma gondii. MBio 8(3). https://doi.org/10.1128/mBio.00375-17
Fox BA, Falla A, Rommereim LM, Tomita T, Gigley JP, Mercier C, Cesbron-Delauw MF, Weiss LM, Bzik DJ (2011) Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection. Eukaryot Cell 10(9):1193–1206. https://doi.org/10.1128/EC.00297-10
Fox BA, Ristuccia JG, Gigley JP, Bzik DJ (2009) Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryot Cell 8(4):520–529. https://doi.org/10.1128/EC.00357-08
Huynh MH, Carruthers VB (2009) Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8(4):530–539. https://doi.org/10.1128/EC.00358-08
Sheiner L, Demerly JL, Poulsen N, Beatty WL, Lucas O, Behnke MS, White MW, Striepen B (2011) A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor. PLoS Pathog 7(12):e1002392. https://doi.org/10.1371/journal.ppat.1002392
Francia ME, Jordan CN, Patel JD, Sheiner L, Demerly JL, Fellows JD, de Leon JC, Morrissette NS, Dubremetz JF, Striepen B (2012) Cell division in Apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella. PLoS Biol 10(12):e1001444. https://doi.org/10.1371/journal.pbio.1001444
Sampels V, Hartmann A, Dietrich I, Coppens I, Sheiner L, Striepen B, Herrmann A, Lucius R, Gupta N (2012) Conditional mutagenesis of a novel choline kinase demonstrates plasticity of Phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii. J Biol Chem 287(20):16289–16299. https://doi.org/10.1074/jbc.M112.347138
Jacot D, Daher W, Soldati-Favre D (2013) Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance. EMBO J. https://doi.org/10.1038/emboj.2013.113
Salamun J, Kallio JP, Daher W, Soldati-Favre D, Kursula I (2014) Structure of Toxoplasma gondii coronin, an actin-binding protein that relocalizes to the posterior pole of invasive parasites and contributes to invasion and egress. FASEB J 28(11):4729–4747. https://doi.org/10.1096/fj.14-252569
Graindorge A, Frenal K, Jacot D, Salamun J, Marq JB, Soldati-Favre D (2016) The Conoid associated motor MyoH is indispensable for Toxoplasma gondii entry and exit from host cells. PLoS Pathog 12(1):e1005388. https://doi.org/10.1371/journal.ppat.1005388
Dogga SK, Mukherjee B, Jacot D, Kockmann T, Molino L, Hammoudi PM, Hartkoorn RC, Hehl AB, Soldati-Favre D (2017) A druggable secretory protein maturase of Toxoplasma essential for invasion and egress. Elife 6. https://doi.org/10.7554/eLife.27480
Di Cristina M, Carruthers VB (2018) New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites. Parasitology 145:1119–1126. https://doi.org/10.1017/S003118201800001X
Shen B, Brown KM, Lee TD, Sibley LD (2014) Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. MBio 5(3):e01114-14. https://doi.org/10.1128/mBio.01114-14
Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S (2014) Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9(6):e100450. https://doi.org/10.1371/journal.pone.0100450
Brown KM, Long S, Sibley LD (2018) Conditional knockdown of proteins using Auxin-inducible Degron (AID) fusions in Toxoplasma gondii. Bio Protoc 8(4). https://doi.org/10.21769/BioProtoc.2728
Frenal K, Jacot D, Hammoudi PM, Graindorge A, Maco B, Soldati-Favre D (2017) Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii. Nat Commun 8:15710. https://doi.org/10.1038/ncomms15710
Jacot D, Tosetti N, Pires I, Stock J, Graindorge A, Hung YF, Han H, Tewari R, Kursula I, Soldati-Favre D (2016) An Apicomplexan actin-binding protein serves as a connector and lipid sensor to coordinate motility and invasion. Cell Host Microbe 20(6):731–743. https://doi.org/10.1016/j.chom.2016.10.020
Jia Y, Marq JB, Bisio H, Jacot D, Mueller C, Yu L, Choudhary J, Brochet M, Soldati-Favre D (2017) Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii. EMBO J 36(21):3250–3267. https://doi.org/10.15252/embj.201796794
Bullen HE, Jia Y, Yamaryo-Botte Y, Bisio H, Zhang O, Jemelin NK, Marq JB, Carruthers V, Botte CY, Soldati-Favre D (2016) Phosphatidic acid-mediated signaling regulates Microneme secretion in Toxoplasma. Cell Host Microbe 19(3):349–360. https://doi.org/10.1016/j.chom.2016.02.006
Donald RG, Carter D, Ullman B, Roos DS (1996) Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J Biol Chem 271(24):14010–14019
Gras S, Jackson A, Woods S, Pall G, Whitelaw J, Leung JM, Ward GE, Roberts CW, Meissner M (2017) Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo. Wellcome Open Res 2:32. https://doi.org/10.12688/wellcomeopenres.11594.2
Kim K, Weiss LM (2004) Toxoplasma gondii: the model apicomplexan. Int J Parasitol 34(3):423–432. https://doi.org/10.1016/j.ijpara.2003.12.009
Pino P, Sebastian S, Kim EA, Bush E, Brochet M, Volkmann K, Kozlowski E, Llinas M, Billker O, Soldati-Favre D (2012) A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host Microbe 12(6):824–834. https://doi.org/10.1016/j.chom.2012.10.016
Plattner F, Yarovinsky F, Romero S, Didry D, Carlier MF, Sher A, Soldati-Favre D (2008) Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response. Cell Host Microbe 3(2):77–87. https://doi.org/10.1016/j.chom.2008.01.001
Herm-Gotz A, Weiss S, Stratmann R, Fujita-Becker S, Ruff C, Meyhofer E, Soldati T, Manstein DJ, Geeves MA, Soldati D (2002) Toxoplasma gondii myosin a and its light chain: a fast, single-headed, plus-end-directed motor. EMBO J 21(9):2149–2158. https://doi.org/10.1093/emboj/21.9.2149
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. https://doi.org/10.1038/nprot.2013.143
Acknowledgments
The author would like to thank Nicolò Tosetti, Aarti Krishnan, and Hung Ryan Vuong for careful reading of the manuscript.
This research was supported by the Swiss National Science Foundation 310030B_166678.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jacot, D., Soldati-Favre, D. (2020). CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii. In: Tonkin, C. (eds) Toxoplasma gondii. Methods in Molecular Biology, vol 2071. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9857-9_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9857-9_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9856-2
Online ISBN: 978-1-4939-9857-9
eBook Packages: Springer Protocols