Advertisement

Phytochromes pp 237-263 | Cite as

CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes

  • Anna Lena Ermert
  • Fabien NoguéEmail author
  • Fabian Stahl
  • Tanja Gans
  • Jon HughesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2026)

Abstract

Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.

Keywords

Physcomitrella patens Phytochrome Gene knockout CRISPR/Cas9 Homologous recombination Nonhomologous end joining 

Notes

Acknowledgments

This work was supported by DFG grant Hu702/5 to JH and by French ANR grant ANR11-BTBR-0001-GENIUS to NF. The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). We thank Pierre François Perroud for his expertise and advice in the context of moss transformation.

References

  1. 1.
    Cove DJ (2000) The generation and modification of cell polarity. J Exp Bot 51:831–838CrossRefGoogle Scholar
  2. 2.
    Jenkins GI, Cove DJ (1983) Light requirements for regeneration of protoplasts of the moss Physcomitrella patens. Planta 157:39–45CrossRefGoogle Scholar
  3. 3.
    Ashton NW, Schulze A, Hall P, Bandurski RS (1985) Estimation of indole-3-acetic acid in gametophytes of the moss, Physcomitrella patens. Planta 164:142–144CrossRefGoogle Scholar
  4. 4.
    Jenkins GI, Cove DJ (1983) Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens: responses of mutant strains. Planta 159:432–438CrossRefGoogle Scholar
  5. 5.
    Jenkins GI, Cove DJ (1983) Phototropism and polarotropism of primary chloronemata of the moss Physcomitrella patens. responses of the wild-type. Planta 158:357–364Google Scholar
  6. 5.
    Jenkins GI, Courtice GR, Cove DJ (1986) Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens. Plant Cell Environ 9:637–644CrossRefGoogle Scholar
  7. 6.
    Schaefer DG, Zrÿd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–1206CrossRefGoogle Scholar
  8. 7.
    Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) Isolation of DNA, RNA, and protein from the moss Physcomitrella patens gametophytes. Cold Spring Harb Protoc 2009(2):pdb.prot5146.  https://doi.org/10.1101/pdb.prot5146CrossRefPubMedGoogle Scholar
  9. 8.
    Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) Transformation of moss Physcomitrella patens gametophytes using a biolistic projectile delivery system. Cold Spring Harb Protoc 2009(2):pdb.prot5145.  https://doi.org/10.1101/pdb.prot5145CrossRefPubMedGoogle Scholar
  10. 9.
    Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) Transformation of the moss Physcomitrella patens using T-DNA mutagenesis. Cold Spring Harb Protoc 2009(2):pdb.prot5144.  https://doi.org/10.1101/pdb.prot5144CrossRefPubMedGoogle Scholar
  11. 10.
    Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) Transformation of the moss Physcomitrella patens using direct DNA uptake by protoplasts. Cold Spring Harb Protoc 2009(2):pdb.prot5143.  https://doi.org/10.1101/pdb.prot5143CrossRefPubMedGoogle Scholar
  12. 11.
    Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) Isolation and regeneration of protoplasts of the moss Physcomitrella patens. Cold Spring Harb Protoc 2009(2):pdb.prot5140.  https://doi.org/10.1101/pdb.prot5140CrossRefPubMedGoogle Scholar
  13. 12.
    Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano RS (2009) Culturing the moss Physcomitrella patens. Cold Spring Harb Protoc 2009(2):pdb.prot5136.  https://doi.org/10.1101/pdb.prot5136CrossRefPubMedGoogle Scholar
  14. 13.
    Rensing SA, Lang D, Zimmer AD et al (2008) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319:64–69CrossRefGoogle Scholar
  15. 14.
    Cove DJ, Schild A, Ashton NW, Hartmann E (1978) Genetic and physiological studies of the effect of light on the development of the moss, Physcomitrella patens. Photochem Photobiol 27:249–254CrossRefGoogle Scholar
  16. 15.
    Possart A, Hiltbrunner A (2013) An evolutionarily conserved signaling mechanism mediates far-red light responses in land plants. Plant Cell 25:102–114CrossRefGoogle Scholar
  17. 16.
    Chen Y-R, Su Y, Tu S-L (2012) Distinct phytochrome actions in nonvascular plants revealed by targeted inactivation of phytobilin biosynthesis. Proc Natl Acad Sci U S A 109:8310–8315CrossRefGoogle Scholar
  18. 17.
    Yamawaki S, Yamashino T, Nakanishi H, Mizuno T (2011) Functional characterization of HY5 homolog genes involved in early light-signaling in Physcomitrella patens. Biosci Biotechnol Biochem 75:1533–1539CrossRefGoogle Scholar
  19. 18.
    Possart A, Xu T, Paik I et al (2017) Characterization of phytochrome interacting factors from the moss Physcomitrella patens illustrates conservation of phytochrome signaling modules in land plants. Plant Cell 29:310–330CrossRefGoogle Scholar
  20. 19.
    Xu T, Hiltbrunner A (2017) PHYTOCHROME INTERACTING FACTORs from Physcomitrella patens are active in Arabidopsis and complement the pif quadruple mutant. Plant Signal Behav 12:e1388975CrossRefGoogle Scholar
  21. 20.
    Cove D, Knight C (1987) Gravitropism and phototropism in the moss, Physcomitrella patens. In: Developmental mutants in higher plants. Cambridge University Press, London, pp 181–196Google Scholar
  22. 21.
    Mittmann F, Brücker G, Zeidler M, Repp A, Abts T, Hartmann E, Hughes J (2004) Targeted knockout in Physcomitrella reveals direct actions of phytochrome in the cytoplasm. Proc Natl Acad Sci U S A 101:13939–13944CrossRefGoogle Scholar
  23. 22.
    Kadota A, Sato Y, Wada M (2000) Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor. Planta 210:932–937CrossRefGoogle Scholar
  24. 23.
    Sato Y, Wada M, Kadota A (2001) Choice of tracks, microtubules and/or actin filaments for chloroplast photo-movement is differentially controlled by phytochrome and a blue light receptor. J Cell Sci 114:269–279PubMedGoogle Scholar
  25. 24.
    Uenaka H, Kadota A (2007) Functional analyses of the Physcomitrella patens phytochromes in regulating chloroplast avoidance movement. Plant J 51:1050–1061CrossRefGoogle Scholar
  26. 25.
    Hughes J (2013) Phytochrome cytoplasmic signaling. Annu Rev Plant Biol 64:377–402CrossRefGoogle Scholar
  27. 26.
    Mittmann F, Dienstbach S, Weisert A, Forreiter C (2009) Analysis of the phytochrome gene family in Ceratodon purpureus by gene targeting reveals the primary phytochrome responsible for photo- and polarotropism. Planta 230:27–37CrossRefGoogle Scholar
  28. 27.
    Li F-W, Melkonian M, Rothfels CJ, Villarreal JC, Stevenson DW, Graham SW, Wong GK-S, Pryer KM, Mathews S (2015) Phytochrome diversity in green plants and the origin of canonical plant phytochromes. Nat Commun 6:7852CrossRefGoogle Scholar
  29. 28.
    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
  30. 29.
    Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34:933–941CrossRefGoogle Scholar
  31. 30.
    Puchta H (2017) Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol 36:1–8CrossRefGoogle Scholar
  32. 31.
    Lopez-Obando M, Hoffmann B, Géry C, Guyon-Debast A, Téoulé E, Rameau C, Bonhomme S, Nogué F (2016) Simple and efficient targeting of multiple genes through CRISPR-Cas9 in Physcomitrella patens. G3 (Bethesda) 6:3647–3653CrossRefGoogle Scholar
  33. 32.
    Nomura T, Sakurai T, Osakabe Y, Osakabe K, Sakakibara H (2016) Efficient and heritable targeted mutagenesis in mosses using the CRISPR/Cas9 system. Plant Cell Physiol 57:2600–2610CrossRefGoogle Scholar
  34. 33.
    Collonnier C, Epert A, Mara K, Maclot F, Guyon-Debast A, Charlot F, White C, Schaefer DG, Nogué F (2017) CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens. Plant Biotechnol J 15:122–131CrossRefGoogle Scholar
  35. 34.
    Collonnier C, Guyon-Debast A, Maclot F, Mara K, Charlot F, Nogué F (2017) Towards mastering CRISPR-induced gene knock-in in plants: survey of key features and focus on the model Physcomitrella patens. Methods 121–122:103–117CrossRefGoogle Scholar
  36. 35.
    Ashton NW, Cove DJ (1977) The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens. Mol Gen Genet 154:87–95CrossRefGoogle Scholar
  37. 36.
    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 17:148CrossRefGoogle Scholar
  38. 37.
    Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832CrossRefGoogle Scholar
  39. 38.
    Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute for Plant PhysiologyJustus Liebig UniversityGiessenGermany
  2. 2.Institut Jean-Pierre BourginINRA, AgroParisTech, CNRS, Université Paris-SaclayVersaillesFrance

Personalised recommendations