CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

Department of Microbiology and Molecular Genetics College of Biological Sciences UC Davis Davis CA USA

Department of Parasitology Faculty of Science Charles University BIOCEV Praha Czech Republic

Institute of Immunology and Microbiology 1st Faculty of Medicine Charles University Prague Czech Republic

Zobrazit více v PubMed

Adam RD. 2021. PubMed DOI PMC

Einarsson E, Ma'ayeh S, Svärd SG. 2016. An up-date on PubMed DOI

Bernander R, Palm JED, Svärd SG. 2001. Genome ploidy in different stages of the PubMed DOI

Tůmová P, Hofštetrová K, Nohýnková E, Hovorka O, Král J. 2007. Cytogenetic evidence for diversity of two nuclei within a single diplomonad cell of PubMed DOI

Tůmová P, Uzlíková M, Jurczyk T, Nohýnková E. 2016. Constitutive aneuploidy and genomic instability in the single-celled eukaryote PubMed DOI PMC

Xu F, Jex A, Svärd SG. 2020. A chromosome-scale reference genome for PubMed DOI PMC

Tůmová P, Dluhošová J, Weisz F, Nohýnková E. 2019. Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated PubMed DOI

Cooper MA, Sterling CR, Gilman RH, Cama V, Ortega Y, Adam RD. 2010. Molecular analysis of household transmission of PubMed DOI PMC

Cooper MA, Adam RD, Worobey M, Sterling CR. 2007. Population genetics provides evidence for recombination in PubMed DOI

Ankarklev J, Lebbad M, Einarsson E, Franzén O, Ahola H, Troell K, Svärd SG. 2018. A novel high-resolution multilocus sequence typing of PubMed DOI

Birky CW. 2010. Giardia sex? Yes, but how and how much? Trends Parasitol. 26, 70-74. ( 10.1016/J.PT.2009.11.007) PubMed DOI

Ankarklev J, Svärd SG, Lebbad M. 2012. Allelic sequence heterozygosity in single PubMed DOI PMC

Poxleitner MK, Carpenter ML, Mancuso JJ, Wang C-JR, Dawson SC, Cande WZ. 2008. Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite PubMed DOI

Yee J, Nash TE. 1995. Transient transfection and expression of firefly luciferase in PubMed DOI PMC

Singer SM, Yee J, Nash TE. 1998. Episomal and integrated maintenance of foreign DNA in PubMed DOI

Sun CH, Chou CF, Tai JH. 1998. Stable DNA transfection of the primitive protozoan pathogen PubMed DOI

Yu DC, Wang AL, Wu CH, Wang CC. 1995. Virus-mediated expression of firefly luciferase in the parasitic protozoan PubMed PMC

Kim J, Park S. 2019. Roles of end-binding 1 protein and gamma-tubulin small complex in cytokinesis and flagella formation of PubMed DOI PMC

Krtková J, Thomas EB, Alas GCM, Schraner EM, Behjatnia HR, Hehl AB, Paredez AR. 2016. Rac regulates PubMed DOI PMC

Hardin WR, Li R, Xu J, Shelton AM, Alas GCM, Minin VN, Paredez AR. 2017. Myosin-independent cytokinesis in PubMed DOI PMC

Kim J, Nagami S, Lee K-H, Park S-J. 2014. Characterization of microtubule-binding and dimerization activity of PubMed DOI PMC

Carpenter ML, Cande WZ. 2009. Using morpholinos for gene knockdown in PubMed DOI PMC

McInally SG, Hagen KD, Nosala C, Williams J, Nguyen K, Booker J, Jones K, Dawson SC. 2019. Robust and stable transcriptional repression in PubMed DOI PMC

Shih H-W, Alas GC, Rydell DS, Zhang B, Hamilton GA, Paredez AR. 2021. An early signaling transcription factor regulates differentiation in DOI

Lin Z-Q, Gan S-W, Tung S-Y, Ho C-C, Su L-H, Sun C-H. 2019. Development of CRISPR/Cas9-mediated gene disruption systems in PubMed DOI PMC

Chen Y-C, Tung S-Y, Huang C-W, Gan S-W, Lin B-C, Chen C-W, Lin Z-Q, Sun C-H, Casamassimi A. 2021. A novel Spo11 homologue functions as a positive regulator in cyst differentiation in PubMed DOI PMC

Ebneter JA, Heusser SD, Schraner EM, Hehl AB, Faso C. 2016. Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in PubMed DOI PMC

Xue C, Greene E. 2021. DNA repair pathway choices in CRISPR-Cas9-mediated genome editing. Trends Genet. TIG 37, 639-656. ( 10.1016/J.TIG.2021.02.008) PubMed DOI PMC

Nenarokova A, Záhonová K, Krasilnikov M, Gahura O, McCulloch R, Zíková A, Yurchenko V, Lukeš J. 2019. Causes and effects of loss of classical nonhomologous end joining pathway in parasitic eukaryotes. mBio 10, e01541-19. ( 10.1128/mBio.01541-19) PubMed DOI PMC

Mowatt M, Luján H, Cotten D, Bowers B, Yee J, Nash T, Stibbs H. 1995. Developmentally regulated expression of a PubMed DOI

Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. 2014. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020-1027. ( 10.1101/gr.171264.113) PubMed DOI PMC

Lauwaet T, et al. 2007. Protein phosphatase 2A plays a crucial role in PubMed DOI PMC

Dolezal P, Smíd O, Rada P, Zubácová Z, Bursać D, Suták R, Nebesárová J, Lithgow T, Tachezy J. 2005. PubMed DOI PMC

Peirasmaki D, Ma'ayeh SY, Xu F, Ferella M, Campos S, Liu J, Svärd SG. 2020. High cysteine membrane proteins (HCMPs) are up-regulated during PubMed DOI PMC

Esvelt KM, Smidler AL, Catteruccia F, Church GM. 2014. Concerning RNA-guided gene drives for the alteration of wild populations. eLife 3, 1-21. ( 10.7554/eLife.03401) PubMed DOI PMC

Dagley MJ, Dolezal P, Likic VA, Smid O, Purcell AW, Buchanan SK, Tachezy J, Lithgow T. 2009. The protein import channel in the outer mitosomal membrane of PubMed DOI PMC

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, 816-821. ( 10.1126/science.1225829) PubMed DOI PMC

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. 2013. Genome engineering using the CRISPR-Cas9 system. Nat. Protocols 8, 2281-2308. ( 10.1038/nprot.2013.143) PubMed DOI PMC

Grzybek M, Golonko A, Górska A, Szczepaniak K, Strachecka A, Lass A, Lisowski P. 2018. The CRISPR/Cas9 system sheds new lights on the biology of protozoan parasites. Appl. Microbiol. Biotechnol. 102, 4629-4640. ( 10.1007/s00253-018-8927-3) PubMed DOI PMC

Broeders M, Herrero-Hernandez P, Ernst MPT, van der Ploeg AT, Pijnappel WWMP. 2020. Sharpening the molecular scissors: advances in gene-editing technology. iScience 23, 100789. ( 10.1016/j.isci.2019.100789) PubMed DOI PMC

Najdrová V, Stairs CW, Vinopalová M, Voleman L, Doležal P. 2020. The evolution of the Puf superfamily of proteins across the tree of eukaryotes. BMC Biol. 18, 1-18. ( 10.1186/s12915-020-00814-3) PubMed DOI PMC

Jiménez-García LF. 2017. The nucleolus of DOI

Markus BM, Bell GW, Lorenzi HA, Lourido S. 2019. Optimizing systems for Cas9 expression in PubMed DOI PMC

Janssen BD, Chen Y-P, Molgora BM, Wang SE, Simoes-Barbosa A, Johnson PJ. 2018. CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite PubMed DOI PMC

Keister DB. 1983. Axenic culture of PubMed DOI

Voleman L, et al. 2017. PubMed DOI PMC

Einarsson E, Troell K, Hoeppner MP, Grabherr M, Ribacke U, Svärd SG. 2016. Coordinated changes in gene expression throughout encystation of PubMed DOI PMC

Morrison HG, et al. 2007. Genomic minimalism in the early diverging intestinal parasite PubMed DOI

Martincová E, et al. 2012. Live imaging of mitosomes and hydrogenosomes by HaloTag technology. PLoS ONE 7, e36314. ( 10.1371/journal.pone.0036314) PubMed DOI PMC

Hughes CS, Moggridge S, Müller T, Sorensen PH, Morin GB, Krijgsveld J. 2019. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protocols 14, 68-85. ( 10.1038/s41596-018-0082-x) PubMed DOI

Rappsilber J, Mann M, Ishihama Y. 2007. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protocols 2, 1896-1906. ( 10.1038/nprot.2007.261) PubMed DOI

Hebert AS, Richards AL, Bailey DJ, Ulbrich A, Coughlin EE, Westphall MS, Coon JJ. 2014. The one hour yeast proteome. Mol. Cell. Proteom. 13, 339-347. ( 10.1074/mcp.M113.034769) PubMed DOI PMC

Cox J, Mann M. 2008. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367-1372. ( 10.1038/nbt.1511) PubMed DOI

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N, Mann M. 2014. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteom. 13, 2513-2526. ( 10.1074/mcp.M113.031591) PubMed DOI PMC

Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, Mann M, Cox J. 2016. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731-740. ( 10.1038/nmeth.3901) PubMed DOI

Tůmová P, Voleman L, Klingl A, Nohýnková E, Wanner G, Doležal P. 2021. Inheritance of the reduced mitochondria of PubMed DOI PMC

Horáčková V, et al. 2022. Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist PubMed DOI PMC

Selective loss of ATP carriers in favour of SLC25A43 orthologues in metamonad mitochondria adapted to anaerobiosis

Mlf mediates proteotoxic response via formation of cellular foci for protein folding and degradation in Giardia

Adaptation of the late ISC pathway in the anaerobic mitochondrial organelles of Giardia intestinalis

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis

Zobrazit více v PubMed

figshare
10.6084/m9.figshare.c.5953852