A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti
Main Article Content
Keywords
Chemical modifications, CRISPR/Cas9, efficiency, parasites, ribonucleoprotein
Abstract
Toxoplasma gondii (T. gondii) and Besnoitia besnoiti (B. besnoiti) are closely related coccidian parasites belonging to the phylum Apicomplexa, which comprises many other important pathogens of humans and livestock. T. gondii is considered a model organism for studying the cell biology of Apicomplexa mainly due to the ease of propagation in diverse host cells and the availability of a wide range of genetic tools. Conversely, B. besnoiti in vitro culture systems currently exist only for the acute phase of infection, and genetic manipulation has proven much more challenging. In recent years, the targeted editing of chromosomal DNA by the programmable CRISPR-associated (Cas)9 enzyme has greatly improved the scope and accuracy of genetic manipulation in T. gondii and related parasites but is still lagging in B. besnoiti. The CRISPR/Cas9 technology enables the introduction of single point and insertion/deletion mutations, precise integration of in-frame epitope tags, and deletions of genes at reduced time and cost compared to previous methods. Current protocols for CRISPR-mediated genome editing in T. gondii rely on either constitutive or transient expression of Cas9 as well as target specific sgRNAs encoded separately or together on transfected plasmid vectors. Constitutively expressed Cas9 carries the risk of toxicity, whilst the transient approach is laborious and error-prone. Here we present a protocol for plasmid vector-independent genome-editing using chemically synthesized and modified sgRNAs. This protocol allows for rapid, efficient, and cost-effective generation of mutant cell lines of T. gondii and B. besnoiti.
Downloads
Download data is not yet available.
Metrics
Metrics Loading ...
References
[1] C. A. Hunter and L. D. Sibley, “Modulation of innate immunity by Toxoplasma gondii virulence effectors,” Nat. Rev. Microbiol., vol. 10, no. 11, pp. 766–778, 2012, doi: 10.1038/nrmicro2858.
[2] G. Álvarez-García, C. F. Frey, L. M. O. Mora, and G. Schares, “A century of bovine besnoitiosis: an unknown disease re-emerging in Europe,” Trends Parasitol., vol. 29, no. 8, pp. 407–415, 2013, doi: https://doi.org/10.1016/j.pt.2013.06.002.
[3] F. Jiang and J. A. Doudna, “CRISPR-Cas9 Structures and Mechanisms,” Annu Rev Biophys, vol. 46, pp. 505–529, 2017, doi: 10.1146/annurev-biophys-062215-010822.
[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science (80-. )., vol. 337, no. 6096, pp. 816–821, 2012, doi: 10.1126/science.1225829.
[5] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014, doi: 10.1038/nature13579.
[6] B. A. Fox, J. G. Ristuccia, J. P. Gigley, and D. J. Bzik, “Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining,” Eukaryot. Cell, vol. 8, no. 4, pp. 520–529, Apr. 2009, doi: 10.1128/EC.00357-08.
[7] B. Shen, K. Brown, S. Long, and L. D. Sibley, “Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii,” in Methods Mol Biol, 2016/10/07., vol. 1498, A. Reeves, Ed. New York, NY: Springer New York, 2017, pp. 79–103.
[8] R. G. Donald and D. S. Roos, “Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria.,” Proc. Natl. Acad. Sci. U. S. A., vol. 90, no. 24, pp. 11703–11707, Dec. 1993, doi: 10.1073/pnas.90.24.11703.
[9] A. Hendel et al., “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells,” Nat. Biotechnol., vol. 33, no. 9, pp. 985–989, 2015, doi: 10.1038/nbt.3290.
[10] S. Kim, D. Kim, S. W. Cho, J. Kim, and J. S. Kim, “Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins,” Genome Res., vol. 24, no. 6, pp. 1012–1019, 2014, doi: 10.1101/gr.171322.113.
[11] D. A. Braasch et al., “RNA Interference in Mammalian Cells by Chemically-Modified RNA †,” Biochemistry, vol. 42, no. 26, pp. 7967–7975, 2003, doi: 10.1021/bi0343774.
[12] R. S. Geary, D. Norris, R. Yu, and C. F. Bennett, “Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides,” Adv. Drug Deliv. Rev., vol. 87, pp. 46–51, 2015, doi: https://doi.org/10.1016/j.addr.2015.01.008.
[13] B. P. Monia, J. F. Johnston, H. Sasmor, and L. L. Cummins, “Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras,” J Biol Chem, vol. 271, no. 24, pp. 14533–14540, 1996, doi: 10.1074/jbc.271.24.14533.
[14] D. E. Ryan et al., “Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs,” Nucleic Acids Res, vol. 46, no. 2, pp. 792–803, 2018, doi: 10.1093/nar/gkx1199.
[15] M. Rahdar, M. A. McMahon, T. P. Prakash, E. E. Swayze, C. F. Bennett, and D. W. Cleveland, “Synthetic CRISPR RNA-Cas9–guided genome editing in human cells,” Proc. Natl. Acad. Sci., vol. 112, no. 51, pp. E7110–E7117, 2015, doi: 10.1073/pnas.1520883112.
[16] A. Mir et al., “Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing,” Nat. Commun., vol. 9, no. 1, p. 2641, 2018, doi: 10.1038/s41467-018-05073-z.
[17] E. K. Brinkman, T. Chen, M. Amendola, and Bas, “Easy quantitative assessment of genome editing by sequence trace decomposition,” Nucleic Acids Res., vol. 42, no. 22, pp. e168–e168, 2014, doi: 10.1093/nar/gku936.
[18] J. G. Doench et al., “Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation,” Nat. Biotechnol., vol. 32, no. 12, pp. 1262–1267, 2014, doi: 10.1038/nbt.3026.
[19] E. Kouranova et al., “CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos,” Hum. Gene Ther., vol. 27, no. 6, pp. 464–475, Jun. 2016, doi: 10.1089/hum.2016.009.
[20] A. Seki and S. Rutz, “Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells,” J. Exp. Med., vol. 215, no. 3, pp. 985–997, 2018, doi: 10.1084/jem.20171626.
[2] G. Álvarez-García, C. F. Frey, L. M. O. Mora, and G. Schares, “A century of bovine besnoitiosis: an unknown disease re-emerging in Europe,” Trends Parasitol., vol. 29, no. 8, pp. 407–415, 2013, doi: https://doi.org/10.1016/j.pt.2013.06.002.
[3] F. Jiang and J. A. Doudna, “CRISPR-Cas9 Structures and Mechanisms,” Annu Rev Biophys, vol. 46, pp. 505–529, 2017, doi: 10.1146/annurev-biophys-062215-010822.
[4] M. Jinek, K. Chylinski, I. Fonfara, M. Hauer, J. A. Doudna, and E. Charpentier, “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science (80-. )., vol. 337, no. 6096, pp. 816–821, 2012, doi: 10.1126/science.1225829.
[5] C. Anders, O. Niewoehner, A. Duerst, and M. Jinek, “Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease,” Nature, vol. 513, no. 7519, pp. 569–573, 2014, doi: 10.1038/nature13579.
[6] B. A. Fox, J. G. Ristuccia, J. P. Gigley, and D. J. Bzik, “Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining,” Eukaryot. Cell, vol. 8, no. 4, pp. 520–529, Apr. 2009, doi: 10.1128/EC.00357-08.
[7] B. Shen, K. Brown, S. Long, and L. D. Sibley, “Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii,” in Methods Mol Biol, 2016/10/07., vol. 1498, A. Reeves, Ed. New York, NY: Springer New York, 2017, pp. 79–103.
[8] R. G. Donald and D. S. Roos, “Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria.,” Proc. Natl. Acad. Sci. U. S. A., vol. 90, no. 24, pp. 11703–11707, Dec. 1993, doi: 10.1073/pnas.90.24.11703.
[9] A. Hendel et al., “Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells,” Nat. Biotechnol., vol. 33, no. 9, pp. 985–989, 2015, doi: 10.1038/nbt.3290.
[10] S. Kim, D. Kim, S. W. Cho, J. Kim, and J. S. Kim, “Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins,” Genome Res., vol. 24, no. 6, pp. 1012–1019, 2014, doi: 10.1101/gr.171322.113.
[11] D. A. Braasch et al., “RNA Interference in Mammalian Cells by Chemically-Modified RNA †,” Biochemistry, vol. 42, no. 26, pp. 7967–7975, 2003, doi: 10.1021/bi0343774.
[12] R. S. Geary, D. Norris, R. Yu, and C. F. Bennett, “Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides,” Adv. Drug Deliv. Rev., vol. 87, pp. 46–51, 2015, doi: https://doi.org/10.1016/j.addr.2015.01.008.
[13] B. P. Monia, J. F. Johnston, H. Sasmor, and L. L. Cummins, “Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras,” J Biol Chem, vol. 271, no. 24, pp. 14533–14540, 1996, doi: 10.1074/jbc.271.24.14533.
[14] D. E. Ryan et al., “Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs,” Nucleic Acids Res, vol. 46, no. 2, pp. 792–803, 2018, doi: 10.1093/nar/gkx1199.
[15] M. Rahdar, M. A. McMahon, T. P. Prakash, E. E. Swayze, C. F. Bennett, and D. W. Cleveland, “Synthetic CRISPR RNA-Cas9–guided genome editing in human cells,” Proc. Natl. Acad. Sci., vol. 112, no. 51, pp. E7110–E7117, 2015, doi: 10.1073/pnas.1520883112.
[16] A. Mir et al., “Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing,” Nat. Commun., vol. 9, no. 1, p. 2641, 2018, doi: 10.1038/s41467-018-05073-z.
[17] E. K. Brinkman, T. Chen, M. Amendola, and Bas, “Easy quantitative assessment of genome editing by sequence trace decomposition,” Nucleic Acids Res., vol. 42, no. 22, pp. e168–e168, 2014, doi: 10.1093/nar/gku936.
[18] J. G. Doench et al., “Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation,” Nat. Biotechnol., vol. 32, no. 12, pp. 1262–1267, 2014, doi: 10.1038/nbt.3026.
[19] E. Kouranova et al., “CRISPRs for Optimal Targeting: Delivery of CRISPR Components as DNA, RNA, and Protein into Cultured Cells and Single-Cell Embryos,” Hum. Gene Ther., vol. 27, no. 6, pp. 464–475, Jun. 2016, doi: 10.1089/hum.2016.009.
[20] A. Seki and S. Rutz, “Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells,” J. Exp. Med., vol. 215, no. 3, pp. 985–997, 2018, doi: 10.1084/jem.20171626.
Article Sidebar
Published
Dec 18, 2020
Article Details
How to Cite
1.
Hehl AB, Winiger RR. A streamlined CRISPR/Cas9 approach for fast genome editing in Toxoplasma gondii and Besnoitia besnoiti. J Biol Methods [Internet]. 2020 Dec. 18 [cited 2024 Apr. 20];7(4):e140. Available from: https://jbmethods.org/index.php/jbm/article/view/343
Issue
Section
Protocols
Authors who publish with JBM agree to the following terms:
- Authors retain copyright and grant JBM right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
