Skip to main content
SpringerLink
Log in

Methods for CRISPR/Cas9 Xenopus tropicalis Tissue-Specific Multiplex Genome Engineering

  • Protocol
  • First Online:
Xenopus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1865))

Abstract

In this chapter, we convey a state-of-the art update to the 2014 Nakayama protocol for CRISPR/Cas9 genome engineering in Xenopus tropicalis (X. tropicalis). We discuss in depth, gRNA design software and rules, gRNA synthesis, and procedures for tissue- and tissue-specific CRISPR/Cas9 genome editing by targeted microinjection in X. tropicalis embryos. We demonstrate the methodology by which any standard equipped Xenopus researcher with microinjection experience can generate F0 CRISPR/Cas9 mediated mosaic mutants (crispants) within one to two work-week(s). The described methodology allows CRISPR/Cas9 efficiencies to be high enough to read out phenotypic consequences, and thus perform gene function analysis, in the F0 crispant. Additionally, we provide the framework for performing multiplex tissue-specific CRISPR/Cas9 experiments generating crispants mosaic mutant in up to four genes simultaneously, which can be of importance for Laevis researchers aiming to target by CRISPR/Cas9 both the S and L homeolog of a gene simultaneously. Finally, we discuss off-target concerns, how to minimize these and ways to rapidly bypass reviewer off-target critique by exploiting the advantages of X. tropicalis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Abu-Daya A, Khokha MK, Zimmerman LB (2012) The hitchhiker’s guide to Xenopus genetics. Genesis 50:164–175. https://doi.org/10.1002/dvg.22007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kok FO, Shin M, Ni C-W et al (2015) Reverse genetic screening reveals poor correlation between Morpholino-induced and mutant phenotypes in Zebrafish. Dev Cell 32:97–108. https://doi.org/10.1016/j.devcel.2014.11.018

    Article  CAS  PubMed  Google Scholar 

  3. Alexandru Dan Corlan (2004) Medline trend: automated yearly statistics of PubMed results for any query. In: Online—own website. http://dan.corlan.net/medline-trend.html

  4. Lansdon LA, Darbro BW, Petrin AL, et al (2017) Identification of Isthmin 1 as a novel Clefting and craniofacial patterning gene in humans. Genetics. doi: https://doi.org/10.1534/genetics.117.300535

    Article  PubMed Central  PubMed  Google Scholar 

  5. Feehan JM, Chiu CN, Stanar P et al (2017) Modeling dominant and recessive forms of retinitis Pigmentosa by editing three rhodopsin-encoding genes in Xenopus Laevis using Crispr/Cas9. Sci Rep 7:6920. https://doi.org/10.1038/s41598-017-07153-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Naert T, Colpaert R, Van Nieuwenhuysen T, et al (2016) CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis. Sci Rep 6. doi: https://doi.org/10.1038/srep35264

  7. Liu Z, Cheng TTK, Shi Z et al (2016) Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos. Cell Biosci 6:22. https://doi.org/10.1186/s13578-016-0088-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. DeLay BD, Corkins ME, Hanania HL, et al (2017) Tissue-specific gene inactivation in Xenopus laevis: knockout of lhx1 in the kidney with CRISPR/Cas9. Genetics. doi: https://doi.org/10.1534/genetics.117.300468

    Article  PubMed Central  PubMed  Google Scholar 

  9. Sakane Y, Iida M, Hasebe T et al (2018) Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas. Biol Open 7. https://doi.org/10.1242/bio.030338

    Article  Google Scholar 

  10. McQueen C, Pownall ME (2017) An analysis of MyoD-dependent transcription using CRISPR/Cas9 gene targeting in Xenopus tropicalis embryos. Mech Dev 146:1–9. https://doi.org/10.1016/j.mod.2017.05.002

    Article  CAS  PubMed  Google Scholar 

  11. MacColl Garfinkel A, Khokha MK (2017) An interspecies heart-to-heart: using Xenopus to uncover the genetic basis of congenital heart disease. Curr Pathobiol Rep 5:187–196. https://doi.org/10.1007/s40139-017-0142-x

    Article  Google Scholar 

  12. Ledford KL, Martinez-De Luna RI, Theisen MA et al (2017) Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation. Dev Biol 426:418–428. https://doi.org/10.1016/j.ydbio.2017.04.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jaffe KM, Grimes DT, Schottenfeld-Roames J et al (2016) c21orf59/kurly controls both cilia motility and polarization. Cell Rep 14:1841–1849. https://doi.org/10.1016/j.celrep.2016.01.069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Nakayama T, Blitz IL, Fish MB et al (2014) Cas9-based genome editing in Xenopus tropicalis. Methods Enzymol 546:355–375. https://doi.org/10.1016/B978-0-12-801185-0.00017-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Jiang F, Doudna JA (2017) CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys 46:505–529. https://doi.org/10.1146/annurev-biophys

    Article  CAS  PubMed  Google Scholar 

  16. Park D-S, Yoon M, Kweon J et al (2017) Targeted base editing via RNA-guided Cytidine Deaminases in Xenopus laevis embryos. Mol Cells 40:823–827. https://doi.org/10.14348/molcells.2017.0262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Aslan Y, Tadjuidje E, Zorn AM, Cha S-W (2017) High-efficiency non-mosaic CRISPR-mediated knock-in and indel mutation in F0 Xenopus. Development 144(15):2852–2858

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Moreno-Mateos MA, Fernandez JP, Rouet R et al (2017) CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun 8:2024. https://doi.org/10.1038/s41467-017-01836-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nakade S, Tsubota T, Sakane Y et al (2014) Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat Commun 5:5560. https://doi.org/10.1038/ncomms6560

    Article  CAS  PubMed  Google Scholar 

  20. Naert T, Van Nieuwenhuysen T, Vleminckx K (2017) TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models. Genesis 55. doi: https://doi.org/10.1002/dvg.23005

    Article  Google Scholar 

  21. Lei Y, Guo X, Liu Y et al (2012) Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs). Proc Natl Acad Sci U S A 109:17484–17489. https://doi.org/10.1073/pnas.1215421109

    Article  PubMed  PubMed Central  Google Scholar 

  22. Guo X, Zhang T, Hu Z et al (2014) Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis. Development 141(3):707–714

    Article  CAS  PubMed  Google Scholar 

  23. Nakayama T, Fish MB, Fisher M et al (2013) Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis 51:835–843. https://doi.org/10.1002/dvg.22720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Blitz IL, Biesinger J, Xie X, Cho KWY (2013) Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system. Genesis 51:827–834. https://doi.org/10.1002/dvg.22719

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Stemmer M, Thumberger T, del Sol Keyer M et al (2015) CCTop: an intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10:e0124633. https://doi.org/10.1371/journal.pone.0124633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Moreno-Mateos MA, Vejnar CE, Beaudoin J-D et al (2015) CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12:982–988. https://doi.org/10.1038/nmeth.3543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Doench JG, Hartenian E, Graham DB et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. Nat Biotechnol 32(12):1262–1267. https://doi.org/10.1038/nbt.3026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Haeussler M, Schönig K, Eckert H et al (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17:148. https://doi.org/10.1186/s13059-016-1012-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Bae S, Park J, Kim J-S (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475. https://doi.org/10.1093/bioinformatics/btu048

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sullender M, Hegde M, Vaimberg EW et al (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191. https://doi.org/10.1038/nbt.3437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Listgarten J, Weinstein M, Kleinstiver BP et al (2018) Prediction of off-target activities for the end-to-end design of CRISPR guide RNAs. Nat Biomed Eng 2:38–47. https://doi.org/10.1038/s41551-017-0178-6

    Article  PubMed  PubMed Central  Google Scholar 

  32. Baker KE, Parker R (2004) Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr Opin Cell Biol 16:293–299. https://doi.org/10.1016/j.ceb.2004.03.003

    Article  CAS  PubMed  Google Scholar 

  33. Mou H, Smith JL, Peng L et al (2017) CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion. Genome Biol 18:108. https://doi.org/10.1186/s13059-017-1237-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kwong LN, Dove WF (2009) APC and its modifiers in colon cancer. Adv Exp Med Biol 656:85–106

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Van Nieuwenhuysen T, Naert T, Tran HT et al (2015) TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis. Oncoscience 2(5):555–566. https://doi.org/10.18632/oncoscience.166

    Article  PubMed  PubMed Central  Google Scholar 

  36. Shi J, Wang E, Milazzo JP et al (2015) Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains. Nat Biotechnol 33:661–667. https://doi.org/10.1038/nbt.3235

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Baklanov MM, Golikova LN, Malygin EG (1996) Effect on DNA transcription of nucleotide sequences upstream to T7 promoter. Nucleic Acids Res 24(18):3659–3660

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  38. Anders C, Jinek M (2014) In vitro enzymology of Cas9. Methods Enzymol 546:1–20. https://doi.org/10.1016/B978-0-12-801185-0.00001-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Shigeta M, Sakane Y, Iida M et al (2016) Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders. Genes Cells 21:755–771. https://doi.org/10.1111/gtc.12379

    Article  CAS  PubMed  Google Scholar 

  40. Bhattacharya D, Marfo CA, Li D et al (2015) CRISPR/Cas9: an inexpensive, efficient loss of function tool to screen human disease genes in Xenopus. Dev Biol 408:196–204. https://doi.org/10.1016/j.ydbio.2015.11.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Moody SA (1987) Fates of the blastomeres of the 32-cell-stage Xenopus embryo. Dev Biol 122:300–319

    Article  CAS  PubMed  Google Scholar 

  42. Burger A, Lindsay H, Felker A et al (2016) Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development 143:2025–2037. https://doi.org/10.1242/dev.134809

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Dr. Tom Van Nieuwenhuysen and Dr. Hong Thi Tran for the collaborative effort in the initial establishment of the CRISPR/Cas9 system within the research unit. Furthermore, we would like to acknowledge the work of Robin Colpaert in comparison of gRNA quantification methodologies and their specific limitations. We would like to acknowledge Dr. Tom van Nieuwenhuysen and Sarah Geurs for the pairwise comparison of Cas9 protein versus mRNA shown in Fig. 3. Finally we would like to thank Marjolein Carron and Dieter Tulkens for critical proof-reading of this manuscript. Research in the authors’ laboratory is supported by the Research Foundation—Flanders (FWO-Vlaanderen) (grants G0A1515N and G029413N), by the Belgian Science Policy (Interuniversity Attraction Poles—IAP7/07) and by the Concerted Research Actions from Ghent University (BOF15/GOA/011). Further support was obtained by the Hercules Foundation, Flanders (grant AUGE/11/14) and the Desmoid Tumor Research Foundation.

Author information

Authors and Affiliations

  1. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Thomas Naert & Kris Vleminckx

  2. Cancer Research Institute Ghent, Ghent, Belgium

    Thomas Naert & Kris Vleminckx

  3. Center for Medical Genetics, Ghent University, Ghent, Belgium

    Kris Vleminckx

Authors
  1. Thomas Naert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kris Vleminckx
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kris Vleminckx .

Editor information

Editors and Affiliations

  1. Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium

    Kris Vleminckx

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Naert, T., Vleminckx, K. (2018). Methods for CRISPR/Cas9 Xenopus tropicalis Tissue-Specific Multiplex Genome Engineering. In: Vleminckx, K. (eds) Xenopus. Methods in Molecular Biology, vol 1865. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8784-9_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8784-9_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8783-2

  • Online ISBN: 978-1-4939-8784-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation